Practical Guide

MICROBIOLOGY
PRACTICAL GUIDE (A)

2010
PRACTICAL I
Introduction
Microorganisms, living creatures too small to be seen with the unaided eye, have a profound
influence on daily life - often beneficial, sometimes harmful.
- bread is made with the help of yeast that ferment sugar and
produce carbon dioxide in the dough before it is baked ;
- cheese, yogurt and sour cream require the presence of microorganisms that sour the milk
from which they are made ;
- some foods are treated to prevent microbial spoilage;
- antibiotics cure such bacterial diseases as step turret bacterial pneumonias, gonorrhea and
syphilis;
- newborn children and adults are immunized against various diseases, including tetanus,
diphtheria, whooping cough, measles and mumps, etc.
- role in biogeochemical cycles
The practicals deepen the knowledge of microorganisms; classical methods such as the use of
aseptic techniques and enrichment culture based techniques; relationship between
microorganisms and their environment; the basis of the phenotypical characterisationof
microorganisms is similarly presented.
The exercises are based on the investigation of representative strains of various bacterial
groups. In designing the exercises, we carefully chose cultures and conditions that minimize
the exposure of students to potentially pathogenic microorganisms and hazardous chemicals.
Laboratory safety
- wear a coat to protect your clothes
- keep your desk free of unnecessary materials at all times and at the end of the period leave it
free of all materials and equipment.
- since many of the microorganisms with which you will be working are potentially
pathogenic, it is imperative to develop aseptic techniques in handling and transferring them.
- avoid any hand - to - mouth operations such as smoking, eating, drinking.
- report immediately all accidents such as cuts, burns or spilled cultures to your instructor;
take every precaution to avoid such accidents.
- some of the chemicals employed in the laboratory can be hazardous if not handled properly,
we have selected the experiments to minimize the use of such substances.
A. Demonstration of microbes in the environment I.
General Rules of sampling: samples always have to be representative! Recording details on
site characteristics and other environmental factors will help when interpreting the sample
results later on. Take extra care to avoid contaminating the sample container and the sample.
1. Sampling the air
Air sampling in the context of microbiological assessment is the collection of airborne
microorganisms. Some may impact product spoilage, product safety and human health.
Collection of vegetative cells and spores may be achieved by passive or active methods.
Passive methods usually involve settle plates (sedimentation) whereas active methods include
impaction and impingement devices. The volume of air for sample collection depends on the
device being used and on the anticipated concentration of the bioaerosol.
Where low concentrations of microbial contaminants are expected, e.g. clean rooms, food
production and operating theatres, impaction methods are generally chosen. In highly
contaminated environments, impaction techniques may 'oversample' even over short
timescales and impingement or filter samples are more appropriate. With the strict adherence
to manufacturer's flow rates, sampling periods, culture media used and device placement,
most techniques should yield comparable results, which are normally expressed in cfu/m
(CFU= colony forming unit).
Fig. 1.1. Cascade impactor

Soil sampling
Soil tests measuremicrobial composition of different soil horizons. The accuracy of a soil test
result is influenced by the laboratory analysis but may be influenced even more by the quality
of the soil sample itself. Sample collection is extremely important in the accuracy and
repeatability of a soil test. Sample handling following collection is also important. A soil
sample which does not represent the area being sampled will be misleading.
Soil physicochemical characteristics influence the level of biomass and the activity of
microorganisms. Seasonal changes in soil moisture, soil temperature and C input from crop
roots, rhizosphere products (i.e. root exudates, mucilage, sloughed cells, etc.), and crop
residues can have a large effect on soil microbial biomass and its activity, which, in turn,
affect the ability of soil to supply nutrients to plants through soil organic matter turnover.
Water sampling (surface water)
Take a labelled sterile sample bottle. Make sure you keep the lid on the bottle
until you are ready to collect the sample.
Hold the sterile bottle in one hand near the base, and then carefully remove and hold the cap
with the other hand. Be careful not to touch the inside of the cap when sampling.
Walk to a location at the water body, which is characteristic for the whole water (e.g. not
polluted by sewage).
Get down on one hand and knee beside the edge of the water body, then immerse the bottle
downwards into the water with continuous motion and direction away from the body but
parallel with the edge of the water body, to a depth of approximately 30 cm below the water
surface and then continue to move the bottle in a horizontal motion until finally removing the
bottle from the water body with the same continued action and motion when full.
Tip enough of the water from the bottle to leave an air space of about 1-2 cm from the rim of
the bottle. This air space is necessary to facilitate mixing of the sample in the laboratory.
Carefully replace the cap immediately and tightly.
Practice:
B. Demonstration of microbes in the environment II.
Demonstration of phototrophic microorganisms
plastic
gum
Air
Aerobic zone
Water (1-2 cm)

Sludge surface
Microaerophilic
zone
\\\\\\\\\\\\\\\\
Anaerobic zone
////////////////
Non-sulfur photosynthetisers (rust red)
"Purple" zone (anaerobic sulfur bacteria)

Chromatium spp.
##
#
###
#
"Green-black" zone
bacteria) Chlorobiaceae
(anaerobic
sulfur
##
2.5 cm sludge riched with CaSO4 and CaCO3
Fig.1.2. Winogradsky column for the enrichment of phototrophic bacteria
The Winogradsky column is a small ecological system in which one can simulate microbial
processes, for example, those occurring in the water and mud of a pond. The Winogradsky
column is simply a clear container or cylinder packed with mud, shredded paper or powdered
cellulose (as a source of energy and carbohydrate) salts of sulfate, carbonate and phosphate;
and water. When sealed and exposed to light, a succession of microorganisms will evolve
according to the amount of oxygen and light available at different points in the column. If the
initial inoculum (the mud) contains the appropriate microbes, the column will enrich for
photosynthetic bacteria, colorless sulfur bacteria and a range of other microorganisms.
Results (after 6 weeks)
At the end of the incubation period, use a Pasteur pipette to sample portions of the column at
various levels. After removing a sample, place a drop on a slide and observe under
microscope in a wet preparation.
Some of the microorganisms you may find in your column are as follows:
1./ Aerobic, sulfide - oxidizing bacteria, for example Thiothrix and Beggiatoa.
2./ Sulfate-reducing microorganisms, such as Desulfovibrio.
3./ Sulfur-oxidizing bacteria that yield H2SO4 and thiosulfate, such as Thiobacillus.
4./ The red sulfide-oxidizing, anaerobic bacteria that grow in illuminated areas, for example
Chromatium
5./ Anaerobic, sulfide-oxidizing bacteria that are all photosynthetic, such as Chlorobium.
Upon microscopic examination, the purple sulfur bacteria can be recognized by their
accumulation of intracellular sulfur granules, the nonsulfur and the green bacteria look much
like spirilla.
C. Demonstration of microbes in the environment III.
Cultivation of microbes from our close environment (air, surfaces). Demonstration of the
effect of hand-wash, etc. The aim of the present practical experiment is to demonstrate
cultivable diversity from our surroundings and to demonstrate how inadequate cultivation
methods may danger work in the laboratory.
1. Cultivation microbes from the air with sedimentation method: exposing media in Petri
dishes to the air for different times (5, 10, 15).
2. Testing surfaces of the laboratory (wall, floor, clothes, etc).
3. Testing human skin:
a. effect of hand washing
Plates must be incubated at 28C for one week.
D. Enrichment of microorganisms
Enrichment media are used to enrich (selectively grow) different microorganisms based on
their specific characteristics (antibiotic resistance, diesel oil degradation capacity, heavy metal
resistance) selective enrichment of bacteria.
Inoculate enrichment media with 1g of soil sample.
1. diesel-oil-containing medium.
2. Medium with cellulose as the sole carbon source .
3. Medium containing HgCl2 in high concentration.
After a week of incubation at 28C, cultures must be spreaded to the adequate Petri dishes.
E. Preparation of sauer kraut
Sauerkraut is a product of fermentation. This completely changes the flavor of the raw
material in addition to giving it long-term preservation qualities.
The sauerkraut fermentation process utilizes the indigenous population of bacteria in the raw
cabbage to produce lactic acid. This produces a low pH environment that allows few, if any,
other bacteria to survive. The lactic acid is also what gives the kraut its characteristic sour
flavor. Salt is added to the raw cabbage to draw out much of the water and to inhibit saltintolerant bacteria. This allows acid-producing bacteria to get a strong foothold and dominate
the population.
The typical routine is to mix the cabbage with salt and then let it sit in a vat or barrel for the 6week fermentation period. It is then ready to eat. It can be taken out as needed but that is were
much of the trouble starts. Highly recommend processing it in a canner or pressure cooker to
prevent further change, which is usually in the direction of spoilage or at least off-flavors.
Mixed acid fermentation : Enterobacteriaceae
Heterolactic fermentation: Leuconostoc mesenteroides
Homolactic fermentation: Lactobacillus brevis, Lactobacillus plantarum
The following simple procedure eliminates all the problems by advancing to the optimum
stage.
o
o
2kgs shredded cabbage

50 g salt (3 tablespoons)
Shred cabbage fine, put it in a large pan. Mix cabbage and salt with your hands. Pack jars
gently with hands till is nearly full. Cover with clean rock or something heavy and close the
bucket. During the curing process, kraut needs daily attention. Remove scum as it forms and
wash and scald rock often to keep it free from scum and mould. At room temperature,
fermentation will be complete in 10 to 12 days. Often there is not enough juice. If this
happens, make thin brine by dissolving 30g of salt in 1L of water.
PRACTICAL II
A. Preparation of microbiological culture media (preparing nutrient agar slants)
The survival and growth of microorganisms depend on available and a favorable
growth environment. Culture media are the nutrient solutions used in laboratories to grow
microorganisms. For the successful culture of a given microorganism it is necessary to
understand its nutritional requirements and then supply it with its essential nutrients in the
proper form and proportions in a culture medium. The general composition of a medium is as
follows:
1.
2.
3.
4.
5.
6.
H-donors and acceptors (approximately 1-15 g/L)

C-source (approximately 1-20 g/L)
N-source (approximately 0,2-2 g/L)
Inorganic nutrients e.g. S, P, (50mg/L)
Trace elements (0,1-1 g/L)
Growth factors (aminoacids, purines, pyrimidines, occasionally 50 mg/L, vitamins
occasionally 0,1-1 mg/L)
7. Solidifying agent (e.g agar 10-20 g/L)
8. Solvent (usually distilled water)
9. Buffers
According to the consistency three types of media are used: liquid, or broth, media;
semisolid media; and solid media. The major difference among them is that solid and
semisolid media contain a solidifying or gelling agent [such as agar, gelatine], whereas a
liquid medium does not.
Liquid media, such as nutrient broth, tryptic soy broth or glucose broth can be used in
studies of growth and metabolism in which it is necessary to have homogenous media
conditions, to follow optical density, and to allow early sampling for analysis of
substrates and metabolic products. Tubes and flasks with liquid cultures can be
incubated with either static or shaken incubation.
Semisolid media can also be used in fermentation studies, in determining bacterial
motility, and in promoting anaerobic growth.
Solid media, such as nutrient agar, are used 1) for the surface growth of
microorganisms in order to observe colony morphology, 2) for pure culture isolation,
3) often in the enumeration and isolation of bacteria from a mixed population by
diluting the original bacteria suspension and spreading a small inoculum over the
surface of the solidified medium and 4) to observe specific biochemical reactions
(extracellular enzymes diffusing away from the colony can be detected as a result of
their action on insoluble substrates present in the agar medium).
Solid media can be poured into either a test tube or Petri dish. If the medium in the test tube is
allowed to harden in a slanted position, the tube is designated an agar slant; if the tube is
allowed to harden in an upright position, the tube is designated an agar deep tube; and if the
agar is poured into a Petri dish, the plate is designated an agar plate.
Media are categorized by their composition:
Chemically defined, or synthetic, media are composed of known quantity and quality
of pure chemicals.
In routine bacteriology laboratory exercises, complex or nonsynthetic media are

employed. These are composed of complex materials rich in vitamins and nutrients,
the chemical composition of which is poorly defined. Three of the most commonly
used components are beef extract, yeast extract and peptone (partially digested
protein).
Media are categorized by their function:
An all-purpose medium, such as Tryptic Soy Agar, supports the growth of most
bacteria cultured in the laboratory. They do not contain any special additives.
Selective media enhance the growth of certain organisms while inhibiting the growth
of others due to the inclusion of particular substrate.
Differential media allow identification of microorganisms usually through the
(visible) physiological reactions unique to those bacteria. The most practical media are
those that both select for and differentiate common pathogens.
Enrichment media allow metabolically fastidious microorganisms to grow because of
the addition of specific growth factors. Enrichment culture is one obtained with the
use of selected media and incubation conditions to isolate the desired microorganisms
from natural samples.
The preparation of media from commercially available dehydrated products is simple
and straightforward. Each bottle of dehydrated medium has instructions for preparation of its
label. For example, to prepare a liter of tryptic soy broth, suspend 30 g of the dehydrated
medium in 1.000 ml distilled water. Mix thoroughly in a 2 liter Erlenmeyer flask [always use
a flask that holds twice the volume of media you are preparing]. Dispense and sterilize for 20
minutes at 121 0C [15 lbs pressure]. As noted, the amount of powder for 1.000 ml of water
will be indicated. In case of preparation of media from a formula pons 500 ml of distilled
water into a 2000ml Erlenmeyer flask. Then measure adequate amount of media components
and dissolve completely in the water in the order of the formula. At the end rins the flask with
the remaining 500 ml water. Mix the medium thoroughly, adjust the pH and sterilize.
If the medium lacks agar, the powder will usually dissolve without heating. If it
contains agar, it is necessary to heat the medium until it starts to boil or even longer in order
to completely dissolve the agar. Most of the exercises you will be doing in this manual will
involve the use of sterile media culture tubes. These tubes must be capped in order to maintain
media sterility. This can be accomplished by using cotton plugs, plastic foam plugs or plastic
or metal caps. All of these caps keep cultures free of contamination while allowing air into the
culture tube, and minimizing evaporation at the same time. It is sometimes desirable to use
screw cap culture tubes. This is especially true when the culture, such as in the case of slants,
may be sealed and stored for long periods. Culture broth can be dispensed with the pipetting
machine, an automatic syringe, or a regular pipette.
Agar deep tubes can be stored after sterilization for use in the preparation of Petri plates when
needed. Some agar deeps may be stored at room temperature for several days before use. If
longer periods of storage are required, they should be placed in the refrigerator in order to
prevent drying of the agar. When Petri plates are needed, the agar deeps are melted either in a
boiling water bath or by bringing them to 121 0C in an autoclave for 30 to 60 seconds, and
then releasing the steam under slow exhaust. After the agar has melted, the pours are
transferred to a 48 to 50 0C water bath and kept there for at least 5 to 10 minutes before use.
The agar deeps should be cooled to about 50 0C before they are used to minimize the amount
of steam condensation on the Petri plate lids after the agar has been poured. Agar does not
solidify until its temperature drops to about 42 0C. When the deeps have reached 50C , one is
taken from the bath and the outside is dried with a paper towel. Its cap is removed and the top
8
is briefly flamed using a Bunsen burner. The agar is immediately poured into a sterile Petri
plate while holding the top carefully above the Petri plate bottom in order to avoid
contamination. Replace the top, allow the agar to cool and harden, and store the Petri plates in
an inverted position.
B. Sterilization of media and equipment
Sterilization is the process of rendering a medium or material free of all forms of life.
There are three basic ways in which sterilization of media and supplies can be achieved. The
most useful approach is autoclaving, in which items are sterilized by exposure to steam at
121 0C and 15 for 15 minutes or longer, depending on the nature of the item. Under these
conditions, microorganisms, even endospores, will not survive longer than about 12 to 13
minutes. Modern autoclaves are designed to ensure that all of the air has been expelled and
only steam is present in the autoclave chamber. They are carefully temperature controlled as
well. Almost all media and anything else that will resist 121 0C and steam can be sterilized
this way.
Often dry glassware, such as pipettes and Petri plates, must be sterilized. Steam tends
to etch glassware and also leaves it damp. Therefore, such items are generally dry - heat
sterilized. The glassware is placed in an electric oven set to operate between 160 and 170 0C.
Since dry heat is not as effective as wet heat, glassware must be kept at this temperature for
about 2 hours or longer. Oven temperature must not rise above 180 0C or any cotton or paper
present will char.
Sometimes media must be made from components that will not withstand heating at
121 0C. Such media can be sterilized by passing it through a (bacteriological) filter, which
physically removes bacteria and larger microorganisms from the solution and thereby
sterilizes them without heat. Scintered glass filters with ultrafine fitted disks [0.9 to 1.4 m
pore size] and Seitz asbestos - pad filter funnels [3 mm thick with 0.1 m pores] are both
quite effective sterilizing solutions. The most useful and popular approach by far is the use of
specially prepared sterile, cellulose- or polycarbonate, etc.-based membranes of the
appropriate pore size. Generally, membranes with 0.22 m pores are employed in
sterilization. A large number of different devices are commercially available for membrane
sterilization of both large and small volumes. For example, one can use a filter flask with
vacuum or syringe with positive pressure to force liquid through a special membrane filter
holder.
Exercise:
a. Measure solid components, dissolve
b. Make final concentration
c. Adjust pH at solidification temperature (in hot form!)
d. Sterilize
e.
Nutrient agar:
- meat extract
3g
- peptone
5g
- agar
18g
- distilled water
1000ml
pH. 7.0, Autoclave for 15 at 121C
1. With different equipments the pressure of vapor, temperature and the impact of time can
be checked permanently.
9
2. Chemical indicators shows wether the temperature and the time period was enough for
sterilization.
3. The use of biological indicators are the most trustworthy methods to calibrate sterilization
equipments. To it standard bacterial spore-preparatios are used (like Bacillus
stearothermophilus). If the efficacy of starilization is insufficient, the test-microbe survives.
Exercise: Controlling the efficacy of sterilization with use of sporepreparation of Bacillus
stearothermophilus strain ATCC 7953.
C. Demonstration of microbes in the environment
Isolation of bacteria, evaluation the CFU (Colony Forming Unit) values from an
environmental sample.
Introduction to cultivation, nutrition and other growth conditions
Viable germ count: the number of viable germs in a culture can be ascertained by
determining the number of colony forming units CFU with the colony counting technique.
(Sometimes microbes clump. A colony may grow from several microbesclustered together.
For this reason, microbiologists often say colonies develop from CFUs rather than from a
single cell.) Between 20 and 200 CFU can be counted on a typical Petri plate. Microbial
cultures of high density must be diluted before they are plated. A known volume [0,1ml] of
these dilutions is plated onto suitable growth medium in the Petri dish. After the plates have
been incubated up to a week, the average number of colonies on plates is determined.
The number of viable microbes per milliliter (or g) of the initial culture (sample) can
be calculated from the average CFUs and the known dilution factor. The viable count is
invariably less than the total cell count because it measures only cells capable of dividing. The
major disadvantages of the colony plating technique are : i./ the incubation period is lengthy,
ii./ sterile media , pipets and plates are required , iii./ sampling and dilution errors occur.
Two methods of this examinations are differentiated:
1) In the pourplate method, a sample from an accurate dilution of microbes/sample is
pipetted onto a Petri - dish, then agar medium is poured over the liquid and mixed.
2) In the spreadplate method, generally 0.1ml of the diluted sample is pipetted onto the
surface of a solidified agar medium and spread with a sterilized, bent, glass rod.
The theory behind the technique of CFU establishes that a single microbe can grow and
become a colony via division. These colonies are clearly different from each other, both
microscopically and macroscopically. This technique allows the user to know how many
CFUs are present per mL in the sample. Therefore, it enables us to know the microbiological
load and the magnitude of the infection in humans and animals, or the degree of
contamination in samples of water, vegetables, soil or fruits and in industrial products and the
equipment.
Quantifying Bacteria by Spread Plate
The number of bacteria in a solution can be readily quantified by using the spread plate
technique. In this technique, the sample is appropriately diluted and a small aliquot is
transferred to an agar plate. The bacteria are then distributed evenly over the surface by a
special streaking technique. After colonies are grown, they are counted and the number of
bacteria in the original sample is calculated.
10
The end point of our analysis is the number of colony forming units per mL (CFU/mL) since
we are counting the number of colonies rather than the actual number of bacteria. CFU/mL is
actually a more useful determination than counting all the bacteria under a microscope,
because in many bacterial populations, a significant number will be dead cells and thus of no
interest.
Diluting the bacteria. Bacteria commonly grow up to densities around 109 CFU/mL,
although the maximum densities vary tremendously depending on the species of bacteria and
the media they are growing in. Therefore, to get readily countable numbers of bacteria, we
have to make a wide range of dilutions and assay all of them with the goal of having one or
two dilutions with countable numbers. We do this by making serial 10-fold dilutions of the
bacteria that cover the entire probable range of concentrations. Then we transfer 0.1 mL of
each dilution to an agar plate, which in effect makes another 10-fold dilution, since the final
unit is CFU/mL and we only streak 0.1 mL.
Inoculating the plate. Streaking in this technique is done using a bent glass rod. 0.1 mL of
bacterial suspension is placed in the center of the plate using a sterile pipette. The glass rod is
sterilized by first dipping it into a 70% alcohol solution and then passing it quickly through
the Bunsen burner flame. The burning alcohol sterilizes the rod at a cooler temperature than
holding the rod in the burner flame, thus reducing the chance of you burning your fingers.
When all the alcohol has burned off and the rod has air-cooled, streak the rod back and forth
across the plate working up and down several times. Unlike streaking for isolation, you want
to backtrack many times in order to distribute the bacteria as evenly as possible. Turn the
plate 90 degrees and repeat the side to side, up and down streaking. Turn the plate 45 degrees
and streak a third time. Do not sterilize the glass rod between plate turnings. Cover the plate
and wait several minutes before turning it upside down for incubation. This will allow the
broth to soak into the plate so the bacteria won't drip onto the plate lid.
Counting bacteria. Colonies are most readily counted using a plate counter. The plate
counter has a light source and a magnifying glass making colonies easier to see. If at all
possible, you don't want to count plates with more than 300 or less than 30 colonies. In the
former case, the colonies , run together, and, in the latter, there are too few to allow
statistically accurate counts. Once you count the colonies, multiply by the appropriate dilution
factor to determine the number of CFU/mL in the original sample.
11
1g
soil
99 mL
0.1 mL
CFU/mL or g: number of colonies on plate x 1/dilution rate

Fig. 2.1. An example of serial dilution and plating
Fig. 2.2. Usage of spreading rod

Exercise:
Make a serial dilution from 1g of soil or 1ml of water sample. Spread onto plates from each
dilution. Thereafter, incubate at 28C for one week. Evaluation of data on the next week.
D. Evaluation of the results of enrichment technique.

E. Measure the pH of sauerkraut
F. Evaluation of the results of Demonstration of microbes in the environment I.
12
PRACTICAL III.
A. Isolation of microbes
Selected colonies will be transferred from Petri dishes into new media to tubes.
B. Colony morphology
Microbes grow on solid media as colonies. A colony is defined as a visible mass of
microorganisms all originating from a single mother cell, therefore a colony constitutes a
clone of bacteria all genetically alike.
Observing colony morphology on inoculated plates
On a given medium, a colonys shape, color, consistency, surface appearance and size - for a
given incubation time - are often characteristic, and these are often of use in the identification
of particular bacterial strains. The full description of a colony can be very detailed.
Thus e.g. the elevation of a colony may be flat, low convex, domed unbonate etc., its edge
maybe entire [circular or unbroken], crenate [scalloped], lobed or fimbriate; its texture may be
butyrous friable or mucoid; its surface may be matt or glossy; it may be whitish or pigmented
or it may contain a dye taken up from the medium, or it may release water soluble pigment
into the medium. The colonies of certain bacteria e.g. Bacillus can migrate across the surface
of a culture plate, the tract of such movement is often marked by lines of bacterial growth
which arise from the cells left behind by the migration colony. An interesting feature of
certain bacterial colonies is the so-called smooth-rough variation. In many types of bacteria,
some type of S-R variation is responsible for a change in the cell-surface composition, which
occurs spontaneously during in vitro or in vivo growth. S-R variation was first recorded in
enterobacteria, in which smooth [glossy] colony may be formed on primary isolation, and
rough [dull] colonies may develop on subcultures.
13
Fig. 3.1. Basic colony morphology types

Identify the following colonial characteristics/culture characteristics:
Whole shape of colony, size of colony (measure with a millimeter ruler), less
than 1mm = punctiform (pin-point).
EDGE/MARGIN OF COLONY: magnified edge shape
CHROMOGENESIS (pigmentation): white, buff, red, purple, etc.
Some pigments are water-soluble, others are not. If you take a large inoculum
and place it in a tube of water or saline, do you see color? Do you see any
pigment if the organism is growing in a broth medium?
OPACITY OF COLONY:
transparent (clear), opaque, translucent (almost clear, but distorted visionlike
looking through frosted glass), iridescent (changing colors in reflected light)
ELEVATION OF COLONY
SURFACE OF COLONY:
smooth, glistening, rough, dull (opposite of glistening), rugose (wrinkled)
CONSISTENCY:
butyrous (buttery), viscid (sticks to loop, hard to get off), brittle/friable (dry,
breaks apart), mucoid
EMULSIFIABILITY OF COLONY:
Is it easy or difficult to emulsify? Does it form a uniform suspension, a
granular suspension, or does not emulsify at all?
ODOR: Absent or present? If it has an odor, what does it smell like?
C. Cultivation of microorganisms, transfer ofmicroorganisms, storage of strains
The practice of transferring microbes from one medium to a new sterile medium is done
as shown on Figure 3.3. in the Practical Instruction Guide. The proper way of sterilizing the
inoculating loop is presented in Figure 3.2. in the Practical Instruction Guide.
14
It is important to keep a Bunsen-burner on throughout the process to prevent contamination

from the air. Always mark each new test-tube properly before transferring microorganisms
(date, sign of the given strain). Never put the caps or cotton plugs down on laboratory
surfaces, keep them in your hand throughout the process.
Fig 3. 2. The proper way of sterilizing the inoculating loop
or
or
or
Fig. 3.3. The practice of transferring microbes from one medium to a new sterile
medium
15
D. Preparation of pure cultures

Streak plate method
Streaks are made across the surface of the agar with a loop full (inoculating loop) of
mixed culture [suspension of Staphylococcus aureus and E. coli or Serratia marcescens and
Micrococcus luteus]. There are reversal different methods for streaking, but in all methods the
objective is to deposit most of the organisms in the first few streaks. Thus, as one continues to
streak the loop back and forth from one section to another of the Petri dish, fewer bacteria will
remain on the loop. If done properly, the last streaks should leave individual bacteria appeare
sufficiently apart from each other so that, after growth, the colonies that have developed from
individual bacteria will be well-separated from each other. A single colony then can be
transferred to a sterile medium, and a pure culture [axenic] will grow.
Strains:
Mixed suspension of type strains
Culture medium:
Nutrient agar plates
Equipment, material: sterile water, pipette, inoculating loop
Fig. 3.4. Preparation of pure cultures

E. Application of REPLICA-technique for the isolation of antibiotic-sensitive or resistant microbes
Using the replica technique, we are preparing copies from the discrete bacterial
colonies that grew on the surface of a Petri-plate. The velvet should be strung onto a
wooden, plastic or metal log. The Petri-dish is then carefully pressed against the surface of
the velvet. The fine filaments of the velvet function as an inoculating loop/needle so that
we are able to transfer all the colonies from the agar plate onto new, sterile agar plates in
just one step. Using selective media (e.g. containing a specific antibiotic) we can get
special metabolic mutants, microbes of distinct abilities.
We are creating replicates from agar plates infected last week (spread plate from soil
and water environmental samples) to agar plates containing a certain antibiotic. Be careful
16
to replicate the plates at the exact same position as the original agar plates. The
incubation of the replicate plates is at 28oC. Evaluation of the results happens the
following week with the absence or presence of the colonies present on the original plates.
Equipment, material: sterile velvet on a replica log, agar plates containing different antibiotics
17
PRACTICAL IV
A.The transfer of the unidentified strain to fresh media (2 copies):
(Everybody gets unidentified bacteria to work with throughout the practical course)
Strains:
Own unindentified strain
Culture medium:
Nutrient agar slants
Equipment, material: Inoculating loop
B.Demonstration and application of different anaerobic culturing techniques
B/1. Deep agar tube containing sodium-thioglycollate
The oldest and most common method of the cultivation of anaerobic bacteria is culturing
them deep in reduced semisolid or solid media. The most important property of these nutrient
media is the suitably low (Eh<-60mV) redox potential. Different indicators within the culture
media serve to test the appropriately low redox potential. These are dyes that are colorless in a
reduced environment. The most suitable indicators are: resazurin and methylene blue, which
show color only when in an oxidized environment (where resazurin is red and methylene blue
is blue). To ensure that oxygen does not get into the culture media either, the inoculated
media is sealed during incubation (melted and heat sterilized vaseline, or vaseline-paraffine 11 mixture is layered on top of the culture media). Semi-solid agar deep tubes are prepared
with a small amount of agar, so that these media are appropriate for the cultivation of
anaerobes without sealing, since agar itself can reduce convection currents, thus reoxidation
as well and agarcolloid applied in small concentration are reductive. To further lower the
redox potential in semisolid media, reductive compounds could be used (sodiumthioglycollate and cystein).
Exercise: Add 1 ml of sediment suspension or water sample to sodium-thioglycollate
containing melted agar deep tubes. Incubate the tubes at 28C for a week. Following
incubation, the amount of colonies formed inside/within the media should be used to estimate
the anaerobe count of the original sample.
Sample:
soil
Culture medium:
Sodium-thioglycollate agar deep tube
Equipment, material: sterile pipette, sterile distilled water
B/2. Cultivation of anaerobic bacteria on Marino-plates
The appropriate method of the cultivation of anaerobes should be chosen with
consideration of the sensitivity of the given organism for the remaining oxygen concentration
within the media or the surrounding atmosphere. For the cultivation of bacteria that are
sensitive to even trace amounts of oxygen (e.g. methanogens and sulphate reducing bacteria),
the best technique is the use of an anaerobic system, filled with an appropriate gas, contacting
the outside world through a sluice gate that removes oxygen entering the system with the use
of a catalyst. Those microbes that are not so sensitive to trace amounts of oxygen and are able
to survive temporarily high oxygen concentrations (e.g. sampling) by forming endospores,
can be cultivated in anaerobic jars or inside usually semisolid media also containing special
reductive agents but incubating at normal conditions. The problem is that colonies forming
inside anaerobic agar deep tubes are difficult to examine and isolate. This problem can be
18
solved by using Marino-plates (better known as Brewer-plates), a combination of a Petri-dish

and anaerobic agar. The Marino-plates provide a bigger surface area to examine the colonyforming properties of anaerobic bacteria and also make the isolation of such bacteria a lot
easier.
Exercise: Prepare a 6-member dilution series (one in ten). If the cultivation of Clostridia is
the aim of the practical, the test tubes containing the dilution series should be put into an 80C
water-bath for 10 minutes to select for endospore forming organisms. The glass Petri-dishes
should be unwrapped under a laminar box. Pipette 100 l from a given dilution onto the
inside surface of the Petri-dish top. Then pour culture media (that has cooled down to
approximately 45C) into the Petri-dish and thoroughly mix the diluted sample with the
nutrient media. Put the bottom of the Petri-dish (with a smaller diameter than the top) onto the
top before the media solidifies; thus creating a thin culture media between the bottom and the
top of the Petri-dish. The Marino-plates then should be wrapped into foil again and incubated
for at least 1-2 days, maybe even for a week depending on the results. Following incubation,
observe the plates where intense bacterial growth can be detected. Examine the individual
colonies under a stereo microscope and try to isolate some of the colonies by making stab
cultures of agar deep tubes.
Equipment, material: sterile Petri-dishes wrapped into foil, warm semisolid
anaerobic agar (e.g. differential RMAC for the cultivation of Clostridia), some sample taken
from anaerobic environment (e.g. an anaerobe layer of lake sediment), 80C water-bath, a
series of test tubes containing 9 ml sterile distilled water for preparing a serial dilution.
B/3. Cultivation of Clostridia in GasPak anaerobic system
The GasPak anaerobic system is a jar that is usually transparent and can be sealed;
contains a palladium catalyst and a disposable H2+CO2 generator and redox indicator.
Cultures are placed into the jar along with an envelope containing chemicals that release
hydrogen and carbon dioxide when activated by water. The envelope includes two tablets, one
that contains NaBH4 that generates hydrogen when reacting with water; the other tablet
contains citric acid and sodium-hydrogen-carbonate that generates CO2 when coming in
contact with water. The CO2 contributes to the support of growth of fastidious anaerobes.
Water is added to the envelope; the jar is then sealed. In the presence of the palladium
catalyst, hydrogen reacts with oxygen to form water. The reaction removes free oxygen from
the inside of the jar.
Equipment, material: anaerobic jar, sterile pipette, spreading glass rod, bismuth-sulfite
agar medium
Exercise: Put the inoculated Petri-dishes with the inoculated surface facing down into the
anaerobic jar (Figure 4.1). Open the redox indicator and place it next to the plates, so that the
indicator strip can be seen from the outside (the indicator strip will turn blue within seconds).
Cut the gas generator envelope with scissors, and pipette 10 ml water through the hole. Close
the jar and screw on the clamp, then put the jar into the thermostat.
19
Figure 4.1 The anaerobic jar system

B/4. Estimation of sulphate reducing bacterial count by MPN method
If sulphate ions are present in the anaerobic environment, the fermentation end product
can be further transformed by the involvement of sulphate-reducing bacteria (SRB). The SRB
is such a specialized group of bacteria whose members proliferate under anaerobic conditions,
and use the energy needed for growth by oxidizing various organic substances with sulphate,
which is then used as the final electron acceptor during anaerobic respiration. The final
product of this respiration is the sulphide ion or hydrogen sulphide (H2S). SRB are more
restricted than fermentative microorganisms in their spectrum of utilized organic
compounds. Most of the fermentative microorganisms are able to transform very complex
organic compounds and polymers while the substrates for SRB are mainly different low
molecular weight organic compounds, the final products of acetogenic fermentation (eg.
lactate, acetate, proprionate). So the SRB are dependent on acid producing bacteria that
provide electron donors to the them .
The SRB is a very diverse group. According to the 16S rDNA sequence analysis, the SRB
seems to be a polyphyletic group. The SRB genera form four main groups: the Gram-negative
mesophile SRB; the Gram-positive endospore forming SRB; thermophile eubacteria and the
thermophile archea SRB. All four groups utilize sulphate as the terminal electron acceptor
during anaerobic respiration.
SRB can be divided into two bigger groups by their metabolic properties. The members of
the first group can oxidize the different organic substrates to acetate (incomplete oxidation
the genera of Desulfovibrio, Desulfotomaculum, Desulfobulbus), while members of the other
group are capable to oxidize these substrates completely to carbon dioxide (the genera of
Desulfobacter, Desulfococcus, Desulfosarcina). Some members of both groups show
hydrogenase activity, thus are able to use hydrogen as electron donor.
Sulphate-reducing bacteria have economic importance since they play an important role in
metal corrosion (their activity can cause the corrosion of gasoline or gas pipes on the inside)
20
and in wastewater treatment processes (part of the biofilm-forming community). Therefore it

is important to study their distribution within a sample.
Equipment, material: the upper 2-5 cm of lake sediment, sterile spoon, sterile 100 ml
Erlenmeyer flask, sterile small test tubes, sterile pipette, well micro-titer plates, PMB broth,
anaerobic chamber
The composition of the PMR medium:
Generalbasal medium:
KH2 PO4
NH4Cl
CaSO4
MgSO4x7H2O
Yeast extract
Ca-Na-lactate
Distilled water
0,5 g
1,0 g
1,0 g
2,0 g
1,0 g
4,0 g
980 ml
pH: 7,0-7,5, sterilization: 1 atm, 121C, 15 min

FeSO4
FeSO4x7H2O
Distilled water
Reductive solution:
Na-thioglycollate
Ascorbic acid
Distilled water
1,1 g
10 ml
0,1 g
0,1 g
10 ml
The preparation of FeSO4 and reductive solutions is done inside the anaerobic
chamber. These solutions are sterilized by filtration and added to the autoclaved basal
medium of approximately 60C.
Exercise: To estimate the bacterial count of the SRB, the culture dependent Most-ProbableNumber (MPN) method is applied. The differential broth Postgates Medium B (PMB) is
used, which is suitable for the cultivation of the two most common genera of SRB: the
Desulfovibrio and the Desulfotomaculum. It is easy to detect the presence and activity of SRB
in the PMB broth, since it contains Fe ions that form black Fe-sulphide precipitate with the
end product of sulphate reduction.
Prepare a 6-member dilution series as follows: Measure 3,0 ml (or 3,0 g) of sample into a
27 ml PMB broth containing 50 ml Erlenmeyer flask. Homogenize the solution with a vortex
at 22C for 5-10 minutes. Pipette 0,3 ml of the obtained suspension into a test tube containing
2,7 ml PMB broth. Mix vigorously, then transfer 0,3 ml suspension again into a new test tube,
and so on. From each dilution (including the original sample), pipette 0,3 ml onto a 96-well
microtiter plate in five parallels (5 test-tube MPN method). The incubation of the microtiter
plates takes place inside the anaerobic chamber of Forma Scientific at 30C for 2 weeks. The
enumeration of total SRB count is estimated by the color change of the PMB nutrient broth
after 1-2 weeks (black precipitate of iron-sulphide). The SRB count is determined by counting
the positive (black colored) wells of the plate and transforming it to the appropriate McCardy
characteristic number to obtainthe MPN value.
21
B/5. Demonstration of the anaerobic chamber (glove-box)

The anaerobic chamber is a device suitable for the cultivation of strictly anaerobic
bacteria. Inside the system, anoxic conditions can be maintained while being able to perform
microbiological operations (microscopy, isolation, inoculation, etc.). Oxygen is removed from
the system with vacuum, then a gas mixture (10% H2, 10% CO2, 80% N2) is introduced into
the system with a slightly positive pressure. The detection of trace amounts of oxygen is
preformed by methylene blue or resazurin redox indicators and the elimination of such
oxygen is completed by a palladium catalyst. Active carbon inside the anaerobic chamber
serves to bind catalyst poisons (e.g. H2S) and other substances that are toxic for bacterial
cells.
C. Preparation of pure cultures II.
Practical C: The purified strains from last weeks practical should be isolated onto fresh,
sterile agar slants.
D. Checking the pH of the sour-cabbage
E. Evaluation of the replica plates from Practical III.
22
PRACTICAL V
A.The transfer of the unidentified strain to fresh media (2 copies):
Strains:
Culture medium:
DEMONSTRATION OF THE EFFECT ENVIRONMENTAL FACTORS ON
BACTERIAL GROWTH
Microbes generally adapt easily to the their natural habitats, however the change in the
physico chemical properties of their environment (e.g. temperature, pH, light, oxygen tension,
osmotic pressure) could greatly affect their life function (e.g. growth rate, pigmentation,
endospore formation).
B.Temperature
B/1. Bacterial growth at different temperatures
Exercise: Transfer the different strains of bacteria (from suspension or agar slants) onto fresh
nutrient agar slants and incubate each strain at 4C, 28C and 37C. Observe the rate of
bacterial growth on the medium after a week-long incubation.
Strains:
Own unidentified strain
B/2. Temperature tolerance

Exercise: Place the already sterilized nutrient medium prepared in big test tubes into water
bath of 60C and 95C. Pipette 0,1-0,1 ml bacterial suspension aseptically into the test tubes,
mix the liquid media with the suspension and incubate at the given temperature for 10
minutes, then pour the contents of each test tube into sterile Petri-dishes. Observe bacterial
growth, then estimate the temperature tolerance of each strain by the number of colonies
formed on the agar plates following incubation at 28C for a week.
Strains:
Materials:
E. coli suspension
Bacillus subtilis suspension
sterile pipette and tips, nutrient agar, water-bath (60C and 95C)
C. Effect of pH on microbial growth

Besides temperature, the hydrogen ion concentration of an organisms environment exerts
the greatest influence on its growth. The concentration of hydrogen ions, which is customarily
designated by the term pH (-log[H+]), effects transport through the cell membrane and limits
the activity of enzymes. As in the case of temperature, an optimum (concentration of
hydrogen ions in which the bacterial growth is most intense) exists for each organism.
Minimum and maximum hydrogen ion concentrations are pH values where an organism still
shows growth. These values are realistic only when other environmental factors remain
constant. If the composition of the medium, incubation temperature, or osmotic pressure is
changed, the pH requirements become different.
23
Exercise: Pipette 0,1-0,1 ml bacterial suspension aseptically into test tubes containing
nutrient broth of different pH values (pH=5, pH=7, pH=9). Estimate the pH tolerance of each
strain by examining the growth rate of the bacteria following incubation at 28C for a week
(observe the turbidity of each broth and compare it with a blank test tube that has not been
inoculated with bacteria).
Strains:
Materials:
E. coli
Staphylococcus aureus
sterile culture media, inoculating loop, sterile water in test tubes
D. The effect of UV radiation on bacterial growth

Most microorganisms are very sensitive to UV radiation. The UV range covers a wide
band of the electromagnetic spectrum (4nm-400nm) but only a narrow range of this spectrum
is responsible for the germicidal effect. There is a very strong bactericidal effect at the
wavelength of 265 nm, due to the damage caused within the DNA replication mechanism.
Usually pigmented and endospore-froming bacteria are most resistant to the effects of
ultraviolet radiation.
Exercise: Expose infected agar plates to UV radiation for different time intervals (10-20-30
minutes). The germicidal effect should be evaluated following incubation at 28C for a week.
Strains:
Materials:
test tubes

sterile nutrient plates, sterile pipette and tips, glass rod, sterile water in
E.Osmotolerance of microorganisms, effect of water activity (aw) on microbial growth

Water activity, (aw). Qualitatively, aw is a measure of unbound, free water in a system,
available to support biological and chemical reactions. Water activity affects microorganisms'
survival and reproduction, enzymes, and chemical reactions. The water activity of a substance
is quantitatively equal to the vapor pressure of the substance divided by the vapor pressure of
pure water (both measured at the same temperature). Water molecules are loosely oriented in
pure liquid water and can easily rearrange. When other substances (solutes) are added to
water, water molecules orient themselves on the surface of the solute and the properties of the
solution change dramatically. The microbial cell must compete with solute molecules for free
water molecules. aw varies very little with temperature over the range of temperatures that
support microbial growth. A solution of pure water has an aw of 1.00. The addition of solute
decreases the aw to less than 1.00.
24
Water Activity of Various NaCl Solutions

Percent NaCl (w/v)
Water Activity (aw)
0.9
0.995
3.5
0.98
7.0
0.96
10.0
0.94
16.0
0.90
22.0
0.86
The aw of a solution may dramatically affect the ability of heat to kill a microbe at a given
temperature. For example, a population of Salmonella typhimurium is reduced tenfold in 0.18
minutes at 60C if the aw of the suspending medium is 0.995. If the aw is lowered to 0.94, 4.3
min are required at 60C to cause the same tenfold reduction. An aw value stated for a microbr
is generally the minimum aw which supports growth. At the minimum aw, growth is usually
minimal, increasing as the aw increases. At aw values below the minimum for growth,
microbes do not necessarily die, although some proportion of the population does die. The
bacteria may remain dormant, but infectious. Most importantly, aw is only one factor, and
other factors (e.g., pH, temperature) must be considered. It is the interaction between factors
that ultimately determines if a microbe will grow or not. A number of food spoilage microbes
can grow within the range 0.8 - 0.6.
Microbe
Water Activity (aw)
Bacteria in general
0.91
Halophiles
0.75
Yeasts in general
0.88
Xerotrophic yeasts
< 0.80
Molds in general
0.80
Xerotrophic molds
0.70
25
The aim of the practical is to compare the osmotolerance of bacteria originating from diverse
environments. The lowering of aw is achieved with the addition of NaCl to the culture media.
Exercise: Inoculate bacterial suspension into culture broth of different salt concentration
values (0%, 5%, 10%). The germicidal effect should be evaluated following incubation at
28C for a week. Estimate the osmotolerance of each strain by examining the growth rate of
the bacteria following incubation at 28C for a week (observe the turbidity of each broth and
compare it with a blank test tube that has not been inoculated with bacteria).
Strains:
Materials:
E. coli
sterile culture broth, inoculating loop
F. Demonstration of the efficacy of different disinfectants

Microbiological laboratories in epidemiological work, medical facilities or the industry
(pharmaceutical companies, food) use disinfectant solutions for preventive, continuous or
terminal disinfection. In the case of an actual epidemiological event (epidemic, accumulation
of contagion), the effectiveness of these disinfectants is randomly inspected. The principle of
efficacy testing is that the disinfectant in question is mixed with the suspension of the test
bacterium, then, after a defined time interval, the disinfectant-microbe suspension is sampled
with an inoculating loop and spread onto the surface of suitable nutrient media. Following the
allotted incubation period, conclusions can be made whether the disinfectant, within the
exposure time interval, killed the test microbe by observing the growth of the test microbe or
the absence of microbial growth.
Exercise: Measure 9-9 ml of each previously diluted disinfectant solution and the control
solution into sterile test tubes and place the test tubes into a water-bath of 25C. Place a sterile
glass rod into the suspension of the test microbe (18-24 h) for 10 minutes. Following the 10minute incubation, take out the glass rod, let the excess suspension flow down the side of the
test tubes. Now place the infected object into the appropriate disinfectant solution and leave
it there for the fixed incubation period of 1, 5, 15, 30, 45 or 60 minutes, then take it out,
immerse in sterile water for 1 minute (to remove the remaining disinfectant from the glass
rod). Inoculate TSA plates with the glass rod and store the inoculated plates at 28C for a
week. Evaluate bacterial growth compared with the control that has not been inoculated on a
scale of five (-, , +, ++, +++).
Strains:
Pseudomonas aeruginosa
Bacillus subtilis
Materials:
1% and 2% Na-hypochlorite-solutions, other disinfectant solutions
Control solution: 0,9% NaCl solution
Equipments:
pipette and tips, vortex, inoculating loop, water-bath, TSA plates
26
No
plates
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
Inoculated
microbe
Inspected material
1% Na-hypochlorite
1% Na-hypochlorite
1% Na-hypochlorite
2% Na-hypochlorite
2% Na-hypochlorite
2% Na-hypochlorite
0,9% NaCl
0,9% NaCl
0,9% NaCl
0,9% NaCl
15
30
45
Pseudomonas aerug.
Bacillus subtilis
Pseudomonas aerug.
Bacillus subtilis
Pseudomonas aerug.
Bacillus subtilis
G. Microbes digesting pectine

Pectinase is a general term for enzymes that break down pectin, a polysaccharide substrate
found in the cell walls of plants. One of the most studied and widely used commercial
pectinases is polygalacturonase. It is useful because pectin is the jelly-like matrix that helps
cement plant cells together and in which other cell wall components, such as cellulose fibrils,
are embedded. Therefore pectinase enzymes are commonly used in processes involving the
degradation of plant materials. Pectinases are actually a mixture of enzymes, which, along
with others such as cellulase, are widely used in the fruit juice industry where they are used to
extract, clarify and modify fruit juices. Pectins are large polysaccharide molecules, made up
(mainly) of chains of several hundred galacturonic acid residues. Enzymes in this pectinase
group include polygalacturonases, pectin methyl esterase and pectin lyases. These pectinase
enzymes act in different ways on the pectins, which are found in the primary cell walls and in
the middle lamella. Pectins are well known also for their ability to form gels. Pectinases are
produced during the natural ripening process of some fruits, where, together with cellulases,
they help to soften their cell walls. These enzymes are also secreted by plant pathogens such
as the fungus Monilinia fructigena and the soft-rot bacterium Erwinia carotovora, as part of
their strategy for penetrating the plant host cell walls. In fact, the products of such enzyme
assaults (oligosaccharins) act as a signal which induces uninfected cells to defend themselves.
Among potato-type plants, the Erwinia species cause significant harm during the cultivation
and storage of the plant. Erwinia carotovora (phytopathogene bacterium) is a Gram negative,
rod shape bacterium, primary agent e.g. of soft rot.
Exercise:
G/1. Action of pectinases during anaerobic digestion - hemp-bath
Make a bunch of hemp (or straw) with cotton yarn. Fill a test tube with water, take one spoon
of soil, then place the hemp into it. Boil it for 3-5 minutes. Use a test tube where the soil is
taken to the tube after the boiling process as controll. After a week of incubation from the
supernatant, spore staining is suggested to see anaerobic spore-forming bacteria (Clostridium
spp.). Effect of boiling: 1. entering the water into plant fibers. 2. kill most non-spore-forming
bacteria from soil.
27
60
G/2. Soft-rot test

After washing, cut slices of potato and carrot and put them into Petri dishes. Inoculate them
with suspension of Erwinia carotovora (100 l respectively). As control, pipette the same
amount of distilled water to one of the slices. Incubate for a week at 28C. Evaluate data on
next practical with Gram staining procedure.
H. Checking the pH of the sour-cabbage
I. Evaluation of the anaerobic cultivation techniques; morphological description of
Clostridium colonies, determination of sulphate reducing bacterial count
28
PRACTICAL VI
A.Transfer of the unknown strain to fresh media:
Strains:
Culture medium:
THE MICROSCOPE
The optical microscope, often referred to as the "light microscope", is a type of microscope
which uses visible light and a system of lenses to magnify images of small samples. Optical
microscopes are the oldest and simplest of microscopes. There are non-optical microscopes,
which require chemical or ion staining of non-living samples, and can magnify exponentially
greater than the optical microscope (e.g. electron microscope).
The components of the microscope
1. ocular lens, or eyepiece 2.

objective turret 3. objective lenses
4. rough adjustment knob 5. fine
adjustment knob 6. object holder
or stage 7. mirror 8. diaphragm 9.
condenser
All optical microscopes share the same basic components:

The eyepiece - a cylinder containing two or more lenses to bring the image to focus
for the eye. The eyepiece is inserted into the top end of the body tube. Eyepieces are
interchangeable and many different eyepieces can be inserted with different degrees of
magnification. Typical magnification values for eyepieces include 5x, 10x and 2x. In
some high-performance microscopes, the optical configuration of the objective lens
and eyepiece are matched to give the best possible optical performance. This occurs
most commonly with apochromatic objectives.
29
The objective lens - a cylinder containing one or more lenses, typically made of glass,
to collect light from the sample. At the lower end of the microscope tube, one or more
objective lenses are screwed into a circular nose piece which may be rotated to select
the required objective lens. Typical magnification values of objective lenses are 4x,
5x, 10x, 20x, 40x, 50x and 100x.
The stage - a platform below the objective which supports the specimen being viewed.
There is a hole in the center of the stage through which light passes to illuminate the
specimen. The stage usually has arms to hold the slides.
The illumination source - below the stage, light is provided and controlled in a variety
of ways. At its simplest, daylight is redirected with a mirror. Most microscopes,
however, have their own controllable light source that is focused through an optical
device called a condenser, with diaphragms and filters available to manage the quality
and intensity of the light.
The complete optical assembly is attached to a rigid arm which in turn is attached to a robust
U shaped foot to provide the necessary stability. The arm is usually able to pivot on its joint
with the foot to allow the viewing angle to be adjusted. Mounted on the arm are controls for
focusing, typically a large knurled wheel to adjust rough focus, together with a smaller
knurled wheel to control fine focus.
Some microscopes have objective lenses, called an oil immersion lens. To use this lens, a
drop of immersion oil is placed on top of the cover slip, and the lens is very carefully lowered
until the front objective element is immersed in the oil film. Such immersion lenses are
designed so that the refractive index of the oil and of the cover slip are closely matched so
that the light is transmitted from the specimen to the outer face of the objective lens with
minimal refraction. An oil immersion lens usually has a magnification of 50 to 100.
The actual power or magnification of an optical microscope is the product of the powers of
the ocular (eyepiece), usually about 10, and the objective lens being used.
Compound optical microscopes can produce a magnified image of a specimen up to 1000
and, at high magnifications, are used to study thin specimens as they have a very limited
depth of field.
How a microscope works

The optical components of a modern microscope are very complex and for a microscope to
work, the entire optical path has to be set up and controlled very accurately. Despite this, the
basic optical principles of a microscope are quite simple.
The objective lens is, at its simplest, a very high-power magnifying glass i.e. a lens with a
very short focal length. This is brought very close to the examined specimen so that the light
from the specimen comes to a focus about 160 mm inside the microscope tube. This creates
an enlarged image of the subject. This image is inverted and can be seen by removing the
eyepiece and placing a piece of tracing paper over the end of the tube. By carefully focusing a
brightly lit specimen, a highly enlarged image can be seen. It is this real image that is viewed
with the eyepiece lens that provides further enlargement.
In most microscopes, the eyepiece is a compound lens, with one component lens near the
front and one near the back of the eyepiece tube. This forms an air-separated couplet. In many
designs, the virtual image comes to a focus between the two lenses of the eyepiece, the first
lens bringing the real image to a focus and the second lens enabling the eye to focus on the
virtual image.
Limitations of light microscopes

At high magnifications with transmitted light, point objects are seen as fuzzy discs surrounded
by diffraction rings. These are called Airy disks. The limit of resolution (Resolving power of
30
a microscope) is therefore taken as the ability to distinguish between two closely spaced Airy
disks (or, in other words the ability of the microscope to reveal adjacent structural detail as
distinct and separate). It is these impacts of diffraction that limit the ability to resolve fine
details. The extent of and magnitude of the diffraction patterns are affected by both the
wavelength of light () and the refractive materials used to manufacture the objective lens and
the numerical aperture (NA or AN) of the objective lens. There is therefore a finite limit
beyond which it is impossible to resolve separate points in the objective field. Assuming that
optical defects in the whole optical set-up are negligible, resolution is indicated with d.
d = 1.22 / 2nxsin
nxsin = numerical aperture (NA)
Usually, a of 550 nm is assumed, corresponding to green light. With air as medium, the
highest practical NA is 0.95, and with oil, up to 1.5. In practice the lowest value of d
obtainable is around 0.2 micrometres or 200 nanometers.
BACTERIAL MORPHOLOGY
The observation of the characteristics of cell morphology is of great importance in the
classification of bacteria with traditional taxonomical methods. These microorganisms cannot
be identified solely by morphological characteristics, since the bacterial cells can only be
assigned to a limited number of categories (Table 6.1). Bacteria are m-sized organisms,
where cell size is an important aspect of a thorough morphological characterization. The size
and shape of the cells are observed by staining. The culturing circumstances, the age of the
culture, the physiological condition of the bacterial cells can each alter the size and shape of
the cells. The shapes of stained bacteria can usually be identified as rods, cocci or spirals. An
average rod shape bacterium is 2-5 m long and 0,5-0,8 m in diameter. The average
diameter of a sphere-shaped bacterium is 0.8 m.
31
Micrococcus
Diplococcus
Streptococcus
Staphylococcus
Tetragenus
Sarcina
COCCUS (SPHERE)
Following cell division, cells separate (singles)
Following cell division, cells remain in pairs
Chain of cocci
Grapelike cluster of cocci
Cell division on 2 planes, cocci in tetrads
Cell division on 3 planes, cocci in aggregates
(packets) of eight
Micrococcus luteus
Neisseria gonorrhoeae
Streptococcus lactis
Planococcus
Sarcina lutea
ROD (BACILLUS)
Shape and size are very variable
long-short, wide-thin, coccoid, irregular
Bacillus
megaterium
Pseudomonas
sp.
Haemophilus influensae
Corynebacterium sp.
CURVED ROD (SPIRAL SHAPE)

Cell with quarter or half a turn
Vibrio
Vibrio comma
Rigid cell wall, moving with flagella, cell with Spirillum volutans
Spirillum
one or more turns
Flexible cell wall, inside flagella, cell with one Treponema palidum
Spirochaeta
or more turns
THREADLIKE
Actinomyces have branching cells, forming Streptomyces

bacterial hyphae and their network (mycelium) Nocardia sp.
sp.
VARIABLE
Intermediate forms
Rhodococcus sp.
Table 6.1. Morphology of bacteria
B. STAINING
Staining is commonly used to facilitate or observe specific organism or intracellular features.
Staining is generally carried out on dead, fixed cells. Extra contrast between different features
in a given specimen may be achieved with counter-staining. In negative staining, the
background is stained or made opaque.
In light microscopy most substances used for staining are themselves colored and they import
their color to the specimen in various ways. Some staining procedures depend on in situ
chemical reaction to produce color. Others involve the deposition of metallic silver
(Spirochaeta spp. ) or of a dye - containing complex (flagella - stain ).
The usefulness of a given stain may depend on its ability to stain some type of cell or cell
component more effectively than others;, thus e.g. certain stains are commonly used for
staining protein, nucleic acid, lipid and cellulose. Whether or not a given dye can obtain a
particular cell or intracellular feature may depend not only on the nature and solubility of the
dye; but on factors such as permeability or integrity of the cell membrane. Another important
factor is pH, thus e.g. a protein pH below its isoelectric point can be stained by an acidic dye
and at a pH above its isoelectric point by a basic dye. The differential staining effect of some
composite stains depends on pH and on the admixture of component dyes in their correct
proportion.
32
B.1. SIMPLE STAINING

Most commonly fixed and stained smears are used for the study of cell morphology,
intracellular constituents and structures. The chemistry of staining is based on the principle
that different charges attract each other, while similar charges repel. In an aqueous
environment, with the pH at 7, the net electrical charge produced by most bacteria is negative.
The dyes that are applied for staining could be acidic dyes, basic dyes and neutral dyes
according to their chemical characteristics. Each dye contains a cation (positive charge) and
an anion (negative charge) where either one could be the chromophore (the part of the
molecule that is brightly colored). Within the bacterial cells, the dyes are bound mostly by the
proteins and nucleic acids (around neutral pH carrying a negative charge). Since acidic dyes
carry a negative charge on their chromophore, the bacterial cell (also negatively charged)
rejects these dyes, so that they can only stain the background of the cells. These so-called
negative dyes include: Eozin Y. Negative staining results from staining with the black
colored India Ink and Negrozin because of the colloidal size of the dye particles which
therefore cannot enter the cell. The chromophore of the basic dyes have a positive charge and
result in an even staining of bacterial cells (positive dyes), since it binds to the cells proteins
and nucleic acids. Basic dyes include safranin (red), methylene blue (blue), crystal violet
(violet), malachite green (green).
Steps in detail:
1. Remove grease from the slides
2. Sign the slide
3. Make smears
4. Fix the smear
5. Stain with basic dye
6. Wash with tap water
7. Dry
8. Microscopy
Exercise: staining own bacterial strain, Micrococcus luteus, Bacillus cereus, Escherichia coli
B.2. DIFFERENTIAL STAINING I.
Gram-staining - an important bacteriological staining procedure discovered empirically in
1884 by Danish scientist Christian Gram. When bacteria are stained with certain basic dyes,
the cells of some species (Gram - negative) can be easily decolorized with organic solvents
such as ethanol or acetone, while cells of Gram-- positive species restrict decolorization. The
ability of bacteria to either retain or lose the stain generally reflects fundamental differences
in the cell wall and is an important taxonomic feature, Gram staining is therefore used as an
initial step in the identification of bacteria. The integrity of cell wall is necessary for Grampositivity, disrupted Gram - positive cells often do not retain the dye. The cells of some
bacteria are strongly Gram-positive when young, but tend to become Gram-negative in ageing
cultures (e.g. Bacillus cereus, Clostridium spp.) this may reflect degenerative changes in the
cell wall. Some bacteria give a Gram-variable reaction, they are sometimes G-positive,
sometimes G-negative; this could reflect e.g. minor variation in staining technique or rather
changes in cell wall thickness, etc.
33
Color changes due to the different steps of Gram staining

Fixed smear
Staining with crystal violet
Treatment with Lugol
Washing with ethanol
Staining with safranine
Gram positive cell

+
+
+
+
Gram negative cell

+
+
-
Steps in detail:
1. Remove grease from the slides
2. Sign the slide
3. Make smears
4. Fix the smear
5. Stain with crystal violet (1)
7. Treatment with Lugol solution (KJ-J)
9. Decolorize with ethanol
11. Stain with safranine (1)
13. Dry it
14. Microscopy
Exercise: staining own bacterial strain, Micrococcus luteus, Bacillus cereus, Escherichia coli
C. BACTERIAL FLAGELLAE
Movement of bacteria with flagellae is observed in liquid media, or in semisolid media while
gliding motility is observed only on solid surfaces.
THE ABILITY OF MOTILITY I.
Indirect method: inoculate tubes of motility medium by stabbing the medium to a depth of
about 5cm. Incubate at the optimum temperature and below. Motile organisms migrate
through the medium which become turbid; growth of non-motile organisms is confined to the
stab.
Motility Test Medium
Bacto Tryptose
10,0 gr.
Sodium Chloride
5,0 gr.
Bacto Agar
5,0 gr.
Destilled Water
1000,0 ml pH = 7,2
Exercise: study own bacterial strain
Fig. 6.1. Motility of bacteria studied by indirect method
34
D. Checking the pH of the sour-cabbage

spreading to Rogosa agar medium
ROGOSA AGAR is used for the isolation, enumeration and identification of Lactobacilli.
This medium is selective, modified by Rogosa to contain high levels of Sodium acetate and
Ammonium citrate at a low pH which inhibits most microorganisms, including Streptococci
and molds and limits swarming but allows for the growth of Lactobacilli. Contains sacharose,
arabinose and dextrose as fermentable carbohydrates, as carbon and energy sources; Tryptone
provides nitrogen, vitamins, minerals and amino acids; Yeast extract provides vitamins,
particularly B-group and other trace elements essential for growth; the Sulfate salts provide
inorganic ions; Sorbitan monooleate is a surfactant; Monopotassium phosphate is the buffer.
Bacteriological agar is the solidifying agent.
Direct inoculation or plate count methodologies can be used.
Inoculate medium and incubate at 35 2C for 24 48 hours.
-
Gram staining from supernatant of the cabbage

F. Movie demonstrating size, shape and motility, etc. of microbes
35
PRACTICAL VII
A. Transfer of the unidentified strain to fresh media (2 copies):
Strains:
Culture medium:
B. Differential staining II.
Acid-fast staining or Ziehl-Neelsen staining - this staining can be used for detection of acidfast bacteria e.g. Mycobacterium spp., Nocardia spp., etc., which is stained with an acid-fast
stain, (mainly carbolfuchsin), cannot be decolorized by mineral acids or by mixtures of acid
and ethanol.
Purpose: The acid-fast stain is a differential stain which distinguishes organisms with waxy
cell walls that can resist decolorization with acid alcohol.
Acid-fast bacteria have a waxy substance called mycolic acid in their cell walls which makes
them impermeable to many staining procedures, including the Gram stain. These bacteria are
termed "acid-fast" because they are able to resist decolorization with acid alcohol. Carbol
fuchsin contains phenol to help solubilize the cell wall. Heat is also applied during the
primary staining to increase stain penetration. All cell types will take up the primary
stain. The cells are then decolorized with acid-alcohol, which decolorizes all cells except the
acid-fast ones. Methylene blue is then applied to counterstain any cells which have been
decolorized. At the end of the staining process, acid-fast cells will be reddish-pink, and nonacid fast cells will be blue. (Note: Acid-fast stains are performed on smears that have been
heat-fixed.)
Steps in detail:
1.
2.
3.
4.
5.
6.
7.
8.
9.
Make smear and fix it

Heat it with carbolfucsin for 10
Wash with water
Decolorize with acid alcohol
Wash with water
Counterstain with methylene blue
Wash with water
Dry
Microscopy
Acid-Fast
Non-AF
Heat + Carbol Fuchsin

Acid alcohol
Methylene blue
Fig. 7.1. Acid fast staining
Staining: Own bacterial strain, Rhodococcus rhodocrous; Staphylococcus aureus

C. Structural staining Schaeffer-Fulton spore staining
Bacterial endospores are highly resistant, thick walled structures formed by vegetative
cells during a process called sporulation. They are highly resistant to radiation, chemical
agents, extremely high temperatures, dessication, and other normally harmful environments.
Several bacterial genera are capable of producing endospores; Bacillus and Clostridium are
the two most common endospore-producing genera. Due to the highly resistant nature of
endospores, it is necessary to steam stain into them. The most common endospore
36
staining technique is the Schaeffer-Fulton method. Once the endospore has absorbed the
stain, it is resistant to decolorization, but the vegetative cell is easily decolorized with water
(leaving the vegetative cells colorless). Finally, the vegetative cells are counterstained with
safranin to aid in their visualization. When viewed under a microscope, the endospores appear
green, while the vegetative cells are red or pink. The steps in the endospore staining technique
are listed below.
1.
2.
3.
4.
5.
6.
7.
Prepare smear, fix with heat

Steam with malachite green
Wash with water
Counterstain with safranin
Wash with water
Dry
Microscopy
Exercise: Bacillus cereus; Saccharomyces cerevisiae; own bacterium

D. Examination of living bacteria [the ability of motility]
Hanging - drop preparation: organisms suspended in a drop of water and placed in the center
of a coverslip. A special slide with a hollow depression in the center is used. The entire slide
with the coverslip is quickly turn over so that the drop of culture is in the center of the
depression. Before observation fix the coverslip with Vaseline/paraffinedrops to the cavity
slide.
- when observing live bacteria, be careful not to confuse motility with Brownian movement
resulting from bombardment with water molecules. In Brownian movement, the organisms all
vibrate at about the same rate and maintain a fairly constant spatial relationship with one
another, whereas bacteria that are definitely motile progress continuously in a given direction.
- motility can be observed most satisfactorily in young cultures [24 or 48 hours] because older
cultures tend to become non-motile . An old culture may become so crowded with inert living
and dead bacteria that it is difficult to find a motile cell. In addition, the production of acid or
other toxic products may result in the loss of bacterial motility in older cultures.
37
PRACTICAL VIII
Strains:
Culture medium:
STUDY OF BACTERIAL ENZYMES
INTRODUCTION
In this exercise microbial physiology, the biochemistry of cells are studied. Microorganisms,
like all living things, may modify their environment to some extent and utilize chemicals in
solution as source of energy and as building blocks for growth and reproduction. With the
help of the biochemical test, we would like to determine which substances the microbe strains
can utilize and what products they produce in the meantime. The chemical end-products of
some of the enzymatic reactions may be measured, or in other cases the disappearance of
certain substances from the medium can be detected. What reactions a species can perform is
determined by its genetic makeup. The DNA carries a pattern for all the enzymes (biological
catalysts) that the microorganisms can produce. Enzymes are substances that alter the rate of
chemical reactions, generally making them happen faster. Enzymes located inside the cells are
called endoenzymes or intracellular enzymes. Those excreted outside of the cells are
exoenzymes or extracellular enzymes. By making a series of different tests, a pattern of
activity can be established (which in turn reflects the enzymatic makeup of the
microorganisms), and this fingerprint aids in the identification and differentiation of the
microorganism from closely related species.
B. EXAMINATION OF RESPIRATORY CHAIN ENZYMES
Energy can be produced by the cell through oxidation which in biological systems is
accomplished primarily by the removal of hydrogen and electrons. Hydrogen is removed from
the substrate (the substance is oxidized) and transported via various respiratory enzymes to a
final hydrogen acceptor. Hydrogen and electron transport supply energy for the cell. The
transfer of hydrogen and the concentration of energy are carried out through a series of
oxidation-reduction reactions.
Biological oxidation occurs with oxygen or some others substances as the final hydrogen
acceptor. Respiration is a biological oxidation occurring with atmospheric oxygen (aerobic
respiration) or inorganic compounds such as nitrates or sulfates (anaerobic respiration).
Fermentation is biological oxidation that occurs when the initial hydrogen donor (usually a
carbohydrate) is broken down and one or more of the dissemination products serve as the final
hydrogen acceptor.
Whether a cell respires or ferments depends generally upon the availability of terminal
electron acceptors and the presence of the enzymes necessary to reduce them. Obligate
aerobes require atmospheric oxygen for the final hydrogen acceptor and will grow only in its
presence.
38
B/1 CATALASE ACTIVITY

Catalase is an enzyme found in most bacteria. It catalyzes the breakdown of hydrogen
peroxide to water and oxygen.
Hydrogen peroxide is toxic to cells. But it is also a common byproduct of metabolic reactions
that take place in the presence of water and oxygen. Therefore most organisms that are able to
survive in an atmosphere containing oxygen produce enzymes to degrade the peroxide. The
major function of catalase within cells is to prevent the accumulation of toxic levels of
hydrogen peroxide formed as a by-product of metabolic processes - primarily that of the
electron transport pathway. Catalase negative organisms tend to be anaerobic. Important
catalasenegative genera are Streptococcus, Leuconostoc, Lactobacillus, Clostridium and
Mycoplasma.
Strains:
Own unidentified strain (24h on agar slant), Proteus vulgaris,
Micrococcus luteus, Bacillus cereus, Escherichia coli
Materials:
3 % H2O2 solution
Exercise: Add a few drops of 3 % H2O2 onto 24h-old cultures. If the strain is catalase
positive, the oxygen gas that has formed can be readily seen as white froth.
B/2. CYTOCHROME OXIDASE
An oxidase is an enzyme that reduces (adds electrons to) oxygen;. Cytochrome oxidase is the
final link in the electron transfer system that provides adenosine triphosphate (ATP) during
aerobic respiration. This enzyme receives electrons and passes them to oxygen, forming
water.
Many bacteria that live in the presence of oxygen produce cytochrome oxidase, but some do
not. Among the Gramnegative rods, for example Pseudomonas, and bacteria closely related
to it, are oxidase producers. Members of the family Enterobacteriaceae are not.
In this exercise, a test strip impregnated with the oxidase reagent is smeared with the bacterial
culture. In a positive test, oxidized cytochrome-e, formed as the result of cytochrome oxidase
activity, oxidiezes p-aminomethylamiline to form a red pigmented product.
Strains:
Own unidentified strain (24h on agar slant) Proteus vulgaris,
Micrococcus luteus, Bacillus cereus, Escherichia coli
Materials:
Oxidase reagent, filter paper, Pt inoculating loop (or glass rod)
Exercise: Add a drop of oxidase reagent onto a filter paper. Let it impregnate the paper. Then
smear some of the test microorganism onto the impregnated surface with the inoculating loop.
(DO NOT use ordinary inoculating loops, since the Fe2+ ions can interfere with the test and
result in a false positive reaction; use one made of Platinum or use a glass rod). If you observe
purplish blue coloration within 30-60 sec., it is considered a positive reaction. Coloration that
forms later than 1 min is considered negative.
39
B/3. METHYLENE BLUE REDUCTION

Some bacteria are able to utilize methylene blue as a terminal electron acceptor when oxygen
is not present. The removal of the oxygen from the broth and the formation of reducing
substances during bacterial metabolism cause the color to disappear because methylene blue
is reduced to the colorless leuco-methylene blue.
Strains:
Materials:
Own unidentified strain (24h on agar slant), Escherichia coli

inoculating loop, methylene blue broth
Exercise: Inoculate a loopful of bacteria aseptically into methylene blue broth and incubate at
28C for a week. The test is positive if the blue color starts to disappear from the bottom of
the tube. If the broth is shaken, then the blue coloration will reappear since oxygen could
dissolve back into the broth.
C. DECOMPOSITION OF BIOPOLYMERS
Milk agar
Proteolytic zone
No degradation
C/1. CASEASE ACTIVITY (casein hydrolysis)

Casein is the primary protein of milk. It exists as a colloidal suspension that gives milk its
opaque cloudiness. The protein is too big to enter the cell, so uptake is only possible
following extracellular hydrolysis. Many bacteria are equipped with enzymes that hydrolyze
this protein into more soluble derivatives. The hydrolyzed protein, and subsequently the milk
itself, become nearly transparent. Protein breakdown, sometimes called peptonization, is a
useful reaction in the identification of bacteria.
Strains:
Materials:
Own unidentified strain (24h on agar slant), M. luteus, B. subtilis

HCl containing HgCl2 solution, skim milk-agar plate
Exercise: Make a spot inoculation (making an X with the loop) on the surface of skim milk
agar plate by placing a loopful of bacteria near one edge of the surface. After incubation,
40
colonies of organisms that are able to digest casein (proteolytic microbes) will be surrounded
by clear zones. Areas in which casein has not been degraded will remain slightly opaque.
In some casesfalse positive reaction could result if the casein is just partly proteolyzed into
para-casein, which also exhibits a clear zone. To eliminate such bad readings of the
resultspour HCl containing HgCl2 solution onto the surface of the milk agar plate. This will
denature proteins along with the para-casein so only areas where full proteolysis has occurred
will remain clear.
C/2. GELATINASE ACTIVITY
The protein gelatin is a water soluble mixture of polypeptides that can be obtained from
connective tissues and tendons of animals by boiling. The substance has a gel forming
characteristic. Many bacteria are capable of producing gelatinase that hydrolyzes gelatin and
breaks down gelatin to utilizable amino acids.
Strains:
Materials:
Own unidentified strain (24h on agar slant)

HCl containing HgCl2 solution, gelatin-agar plate
Exercise: Make a spot inoculation (making an X with the loop) on the surface of gelatin agar
plate by placing a loopful of bacteria near one edge of the surface. After incubation (a week)
the agar plates are flooded with HCl containing HgCl2 solution. Within minutes the HgCl2
denatures proteins, forming an opaque precipitate with gelatin. Clear zones will appear where
the gelatin has been broken down.
C/3. -AMYLASE ACTIVITY
Starch is a polysaccharide that is abundant in nature; a rich source of carbon and energy. It is
a common reserve carbohydrate supplying the nutritional need of plant roots and seeds. But
the polysaccharide is too large to cross bacterial semipermeable cell membranes. Amylases
cleaves large starch molecules into monosaccharide and disaccharide units small enough to
enter into a bacterial cell. There the sugars are degraded by endoenzymes, releasing energy
and carbon.
Strains:
Materials:

Iodine solution, Starch agar plate
Exercise: Make a spot inoculation (making an X with the loop) on the surface of starch agar
plate by placing a loopful of bacteria near one edge of the surface. After incubation (one
week) the agar plates are flooded with iodine solution. Evaluation of the results is based on
the reaction of starch with iodine which forms a blue coloration. Clear zones around the
inoculated area indicate a positive reaction, where amylase has destroyed starch.
C/4. LYPOLYTIC ACTIVITY (TWEEN 80 HYDROLYSIS)
Lipids are found in every kind of cell membrane, and they are common in plant and animal
nutrient storage compounds. The common lipids decomposed by microorganisms are
triglycerids and phospholipids. The triglycerids are esters of glycerol and fatty acids. The
reaction involved in their hydrolysis occurs as follows:
41
O
CH
2
CH
CH OH
2
3H O
R'
2
CH OH
+ R' COOH
R''
CH OH
2
R'' COOH
O
C
RCOOH
O
CH
2
triglyceride
lipase
glycerol
3 fat ty acids
molecules
(Tween 80 is the oleic acid ester of a polyoxyalkylene derivative of sorbitan)

One of the main groups of esterases are the lipases. Most methods described for the disposal
of bulk wastes employ various methods for the removal of fats and greases because their
presence would markedly increase the requirement for available oxygen and in turn slow
down the main process of sewage decomposition.
Strains:
Materials:

Tween 80 agar plate
Exercise: Make a spot inoculation (making an X with the loop) on the surface of Tween 80
nutrient agar plate by placing a loopful of bacteria near one edge of the surface. After
incubation evaluate the results on the basis of the presence or absence of Ca-oleate crystals
near the area of inoculation. In the case of a positive reaction by the hydrolysis of Tween,
oleic acid is freed and the calcium ions present in the media form Ca-oleate precipitation
zones around the inoculation.
C/5. DNASE ACTIVITY
Organisms may be incubated on plates of DNA agar. After incubation flood the plates with
1M-HCl; a positive result shows as a clear zone around the growth the surrounding medium and a negative result of the rest - is opaque.
Strains:
Materials:

1N HCl solution, DNA containing agar plate
Exercise: Make a spot inoculation (making an X with the loop) on the surface of DNA
containing agar plate by placing a loopful of bacteria near one edge of the surface. After
incubation pour 1N HCl solution on the surface of the plates. The acid denatures and
precipitates DNA, so where the reaction is positive, and DNA has been broken down, there
will be clear zones around the inoculated area. The test is negative if there is no clear zone
visible around the spot inoculation.
D. HAEMOLYSIS
The majority of bacterial pathogens produce erythrocyte destroying (haemolysis) enzymes.
There are two types of hemolysis: , and haemolysis. If the erythrocytes are completely
destroyed, the bacterial colonies are surrounded by clear zones; this is the case for
42
hemolysis. In other cases, there is a greenishbrownish zone visible around the inoculated
area, which can be accounted for the partial degradation of red blood cells.
Strains: Escherichia coli, Staphylococcus aureus, Streptococcus faecalis, Streptococcus
sp., Bacillus cereus, Bacillus cereus, Own unidentified strain
Materials: Blood-agar plate, inoculating loop
E. CARBOHYDRATE (MONO AND OLIGOSACCHARIDES) DEGRADATION

E/1. HUGHLEIFSON TEST: GLUCOSE OXIDATIVE AND FERMENTATIVE DEGRADATION
The oxidationfermentation test is used to distinguish between fermentation for which
oxygen is not necessary and respiration for which oxygen is used as terminal electron
acceptor. A semisolid agar medium together with a sugar glucose and bromthymol blue
indicator that changes from blue to yellow when the environment becomes acidic. For each
test, two agar deep tubes are needed: the aerobe tube and the fermentative tube, which is
sealed with a thick layer of paraffin to prevent oxygen from penetrating into the medium.
Strains:
Materials:
Hugh-Leifson semisolid agar deep tube (one aerobe and one anaerobe
(sealed with paraffin), both having a bluish-green colour) per strain, inoculating loop
Exercise: Inoculate both the aerobe and the fermentative tubes with a loopful of the given
bacteria by making a stab inoculation deep inside the agar tubes. When inoculating the
fermentative agar deep, you must stab through the paraffin layer, so be careful because during
the sterilization of the inoculating loop the adhering paraffin might catch fire and squirt.
Incubate the tubes at 28C for a week. Evaluation of the results is as follows:
Fermentation: The fermentation of the glucose starts at the most anaerobic part of the tube
(the bottom) so read the fermentative agar deep tube from the bottom. A yellow coloration at
the bottom of the fermentative tube indicates a week positive reaction. Acid is formed in both
tubes for fermentation.
Oxidation: Aerobic utilization of glucose starts at the most aerobic part of the media (top of
the tube), therefore starting the readings of the oxidative tube from the top. A yellow color in
the upper portion of the tube indicates aerobic utilization of glucose.
E/2. METHYL RED AND VOGESPROSKAUER TESTS
This test is preformed in MRVP Medium containing glucose, peptone, phosphate buffer and
methyl red indicator. This MRVP test is used to identify organisms able to produce acetoin
from the degradation of glucose during a 2,3-butanediol fermentation. Both the MR and VP
tests are especially useful in differentiating between members of the Enterobacteriaceae.
Acetoin
-napthol
KOH
O2
diacetyl-creatin complex
(red colour)
Strains:
Materials:
MRVP Medium, napthol (Barritt reagent A), 40% KOH (Barritt
reagent B), empty test tube, methyl red indicator
43
Exercise: Inoculate a loopful of the test microbe into MR-VP medium. Incubate the tubes at
28C for a week. When evaluating the results, take 1 ml of sample into a new test tube.
After 48 hours of incubation, Barritt's Reagent A (-napthol) and Barritt's Reagent B
(potassium hydroxide) are added to the sample. After shaking the tube gently for aeration,
formation of a red color after approximately 20 min. will indicate a positive VP reaction.
Add 3-4 drops of methyl red indicator solution to the remaining MR-VP medium. A lasting
red coloration is considered to be positive MR reaction.
E/3. AESCULIN HYDROLYSIS
Esculin (-glucose-6,7-dihydroxycoumarin) hydrolysis is a useful test in the differentiation of
both gram-positive and gram-negative bacteria. Hydrolysis is indicated by the production of a
brownish-black colored compound, due to the combination of ferric ions (Fe3+) with the
hydrolysis product esculetin (6,7-dihydroxycoumarin) as indicator.
hydrolysis
-glucose-6,7-dihydroxycoumarin glucose
6,7-dihydroxycoumarin
(Esculetin)

Esculin broth, inoculating loop
Strains:
Materials:
Exercise: Inoculate a loopful of the test microbe into Esculin broth. Incubate the tubes at
28C for a week. A blackish, dark brown colloidal precipitate forms in the test tube if the test
is positive. If there is no precipitate, the test is negative.
F. AMINO ACIDS
F/1. H2S PRODUCTION (CYSTEIN DESULFHYDRASE)
Bacteria that produce the enzyme cystein desulfhydrase are able to strip the amino acid
cystein of both its sulfhydryl (-SH) and amino groups. The reaction yields hydrogen sulfide
(H2S), ammonia ( NH3 ) and pyruvic acid. Hydrogen sulfide reacts with heavy metals such as
lead or iron to form a visible, black precipitate.
HS CH2 CHNH2 COOH
Cystein
H2S + Pb(CH3COO)2
Strains:
Materials:
H2S +NH3+CH3 CO COOH

gas
pyruvic acid
PbS + 2CH3COOH
black precipitate

Nutrient broth, inoculating loop, filter paper impregnated with
Pb(CH3COO)2
Exercise: Inoculate a loopful of the test microbe into cystein containing broth. Place the test
stripe (lead acetate impregnated filter paper) into the tube, so that it does not hang into the
liquid medium and it is fixed between the cap or plug and the side of the test tube. (Use
gloves when handling the filter paper). Incubate the tubes at 28C for a week and examine the
filter paper. The test is positive if the paper turns black, the test is negative if there is no
coloration of the filter paper.
44
F/2. INDOLE
PRODUCTION
Some bacteria hydrolyze tryptophan to pyruvic acid, which is then metabolized in a pathway
similar to the familiar Krebs pathway. A byproduct of that hydrolysis is indole, which is
excreted by the organism. Pure tryptophan is not ordinarily used in the test medium. Instead,
tryptone - a digestion product of certain proteins - is used as the substrate since it contains
considerable amount of tryptophan. The presence or absence of the responsible enzyme,
tryptophanase, is important in the differentiation of enteric bacteria such as Eschericia coli.
NH
+H O
2
*
CH
CH
COOH
2
tryptophan
NH
2
NH
+H O
* 2
CO
+ CH
pyruvic acid
indole
Strains:
Materials:
COOH + NH
3

Nutrient broth, inoculating loop, Kovcs reagent
Exercise: Inoculate peptone broth with the test microbe and incubate the tubes at 28C for a
week. Add 0,2 ml of Kovcs reagent (5% para-dimethyl-amino-benzaldehyde in 75% amyl
alcohol, 25% concentrated hydrochloric acid) to the test tube, mix well and let it rest for about
5 min. A positive test is indicated by a red coloration in the upper alcohol layer. The test is
negative if the upper alcohol layer is yellow.
G. PHOSPHATASE ACTIVITY
This test was used by Barben & Kirby to aid the identification of pathogenic staphylococci ;
they found a high degree of correlation between phosphatase and coagulase production.
phosphatase
Na- phenolphthalein-phosphate
phosphate
+
phenolphthalein
(red in alkaline solution)
Strains:
Materials:

Na- phenolphthalein-phosphate agar plate, inoculating loop, ammonia
Exercise: Inoculate a loopful of the test microbe onto the surface of Na- phenolphthaleinphosphate agar plate and incubate the plates at 28C for a week. Turn the agar plates upsidedown and pipette a drop of ammonia onto the top of the Petri-dish. If the test bacteria is
phosphatase positive, free phenolphthalein liberated by phosphatase reaction reacts with the
ammonia and phosphatase positive colonies become bright pink.If the test is negative, there is
no visible color change.
45
H. Cultivation of antibiotic producer microbe: Starting the shaken cultures of Streptomyces

strains
46
PRACTICAL IX.
Strains:
Culture medium:
B.Determinationofthelysosimecontentofanegg
Lysosime is an enzyme responsible for the digestion of murein of bacteria, produced by
several organisms (plants, animal tissues, bacteriophages, bacteria etc).
Test will be evaluated by the decrease of the optical density of Micrococcus luteus
suspension.
1. Making suspension from M. luteus (24-hour culture) in 5 ml phosphate buffer.
2. Egg white is diluted to 1000x with phosphate buffer.
3. Standard: lysosime solution (4g/ml)
4. Making a dilution from samples as follows:
Half test tube
Buffer (ml)
3,5
3,75 3,88 3,94
sample (ml)a
0,5
0,25 0,12 0,06
0,3
0,3
0,3
0,3
0,3
M. luteus suspension (ml)
0,3
0,3
Gg white OD (30 min.)

Standard OD (30 min.)
a
egg white or lysosime solution
5. Pipette 0.3 ml M. luteus suspension to the dilution steps in 1minute intervalls and mix
it.
6. Incubate at room temperature for 30 minutes.
7. After incubation, determine the optical density of solutions at 520 nm wavelength.
C. Antimicrobial gs
These are chemicals used to treat diseases; they include antibiotics, a group of compounds
originally produced by metabolic reactions of bacteria and fungi that prevent the
multiplication of other microbes. Most antibiotics are produced by bacteria (genera
Streptomyces and Bacillus and fungal genus Penicillium). Some of those are
chemotherapeutic agens - may be defined as chemicals that, at concentrations toleraded by the
host, can interfire directly with the proliferation of pathogenic micro-organisms. Thus the
essential feature is one of selective toxicity. Some drugs are bacteriostatic, the inhibition of
growth being reserved when the drug is removed, others are bactericidal, exerting an
irreversible lethal effect. The distinction between bactericidal and bacteriostatic action is
however not absolute, since some normally bactericidal agents appear to be bacteriostatic at
relatively low concentrations.
47
Antibiotics: were originally defined by Waksman (1945) as chemical substances produced

by microorganisms which possess the ability to kill or to inhibit the growth of bacteria and
other microoganisms. Most of these compounds act by inhibiting the formation of a particular
type of macromolecule in the microbial cell. For example, penicillus and cefalosporius inhibit
peptidoglycan synthesis; streptomycin, chloramphenicol and the tertacyclines interfere with
protein biosynthesis, refampicin and actinomycin D prevent nucleic acid synthesis, polymyxin
B, valinomycin and gramicidin A inhibit the cell membrane function.
C/1.TheuseofResistestdiscsKirbyBauerTechnique:
In this method a culture of known concentration is spreaded on an appropriate medium
(Mueller-Hinton medium is widely used because it is an appropriate growth medium for most
bacterial pathogens and it contains very few substances that inactivate antibiotics).
Filter paper discs containing predetermined concentrations of an antimicrobial are placed on
the seeded agar plate, with equal spacing between discs (Generally 4 discs / Petri dish).
During incubation, the agent diffuses out of the disc, creating a concentration gradient that
decreases according to distance away from the disc. After the incubation the organisms,
sensitivity is measured on the basis of the size of the zone of inhibition (no growth) arround
each disc. This measure is compared to values on a standard, and it indicates whetwer the
microorganism is resistant, intermediate or sensitive to the agar. Sensitive means that the
organism is inhibited by clinically attained concentrations of the antimicrobial; resistant
means that the organisms is not inhibited; intermediate meant that special considerations are
to be followed if the antibiotic is to be used.
R : resistant zone of inhibition with a diameter equal to or less than inner circle.
I : intermediate zone of inhibition with a diameter greater than R.
S : sensitive zone of inhibition with a diameter greater than outer circle .
48
Fig. 9.1 Determination of sensitivity to antibiotics

The Kirby-Bauer method: i./ standardized the concentration of antibiotics to be tested ;
ii./ standardized the incubation time ;
iii./ standardized the nature of inoculum
iv./ standardized measurement of the inhibition zone.
Antibacterials
1./ Alexander Fleming discovered penicillin in 1929, however, it was not tested in a human
until 1941, when Florly and Clain purified enough penicillin to carry out clinical trials.
Penicillin G became widely available after War World II. The development of the
semisynthetic penicillins had to wait until the microbial production of 6-aminopenicillinic
acid in the late 1950s.
2./ The range of the microbes affected by an antimicrobial agent is its spectrum, antibiotics
are either broad spectrum or narrow spectrum antimicrobials.
3./ Antibiotics inhibit the growth of (bacteriostatic) or kill (bactericidal) microorganisms by
interfering with protein synthesis, nucleic acid synthesis, cell-wall synthesis, or cellmembrane function.
4./ Bacteria acquire resistance to antibiotics when a structure is altered so as to prevent
antibiotic binding or the cell acquires genes (plasmid) encoding enzymes that inactivate the
antibiotic.
C/2. In vitro synergy between combinations of antimicrobial agents
The addition of two or more antimicrobial agents to a microbial population sensitive to each
of the individual compounds may have various outcomes. The overall inhibitory effect may
be insignificantly different from that of the sum of the individual agents (indifference), it may
show enhancement over that of the individual agents (synergy), or it may be considerably
lower than that anticipated from the individual responses (antagonism). For example,
combinations Trimethoprim and sulfonamides are murkedly synergistic to a variety of
microorganisms, while polymyxin and sulphonamides show synergy with some Proteus
species. Antagonism between nalidixic acid and nitrofurantoin has been reported for some
Proteus isolates and Staphylococous aureus sensitivity to lincomycin is antagonized by
erythromycin.
49
The technique used in this experiment involves placing sensitivity disc soaked in solutions of
different antimicrobial agents onto seeded agar plates such that they overlap, and examining
the nature of any areas of growth inhibition surrounding the intersection after incubation.
C. Antimicrobial compounds of plants (chamomilla, horse radish, onion, etc.)
Many plants contain chemical compounds that inhibit the growth of microorganisms and this
may play an important part in their resistance to pathogens. Different plants produce different
compounds which vary in their antimicrobial action and microorganisms differ in their
sensitivity to the compounds. Garlic is a good example of one of the many plants that contain
such antimicrobial compounds.
The technique used in this case involves placing slices of garlic onto the seeded agar plates
and examining the nature of any areas of growth inhibition surrounding the garlic slice after
the incubation.
Exercise:
1. Spread Petri dish media with the adequate bacterial suspension
2. Place onion/garlic to it
3. OR: make a hole on the spreaded Petri dish and fill it with the adequate plant extract
4. Incubation
D.Detectionofantagonismincrossstreakexperiments
1. antimicrobial producer strain
2. test organisms
E. Screening the filtrate of Streptomyces strain

Microorganisms produce several primary and secondary metabolites. Some of them have
antimicrobial activity.
Testorganism: Staphylococcus aureus
1. Shake a given Streptomyces strain for 1 week, then filtrate it sterile.
2. OD of test organism must be 0.3 at 660 nm.
3. Add 1 ml S. aureus suspension to 100 ml melted (50C) agar media, pour it to Petri
dishes and solidify it.
4. Make a hole in the infected plates, pipette to the holes the filtrate of Streptomyces.
5. Inhibition zones can be measured after 24 hours.
50
F. Effect of heavy metals to bacteria

Heavy metals (Ag, Cu, Ni, etc) are toxic for microorganisms already in low concentrations
(denaturation of proteins). In the present exercise, ions of the given heavy metal will diffuse
into the medium and thus produce an inhibition zone.
Exercise:
1. Spread Petri dish media with the adequate bacterial suspension
2. Place a small piece of Cu plate on it
3. OR make hole on the spreaded Petri plate and fill it with the adequate solution
(CuSO4)
Strains: Escherichia coli
51
PRACTICAL X
Coliforms are good indicator organisms. They are not generally pathogenic, their presence
shows that faecal contamination of water has occurred. Coliforms in the hygienic practice are
defined as those facultative anaerobic, Gram-negative, nonendospore-forming, rod-shaped
bacteria that ferment lactose, produce acid and gas within 48 hours at 37C. Coliforms are
members of the family Enterobacteriaceae (e.g. E. coli, Enterobacter aerogenes, Klebsiella
pneumoniae). The values of coli-count and the coli-titer are used for the quantitative
characterization of coliform organisms in a sample.
Coli-titer is the smallest amount of water, from which coliform organisms can still be
cultivated.
Coli-count (Coli-index) is the number of coliform bacteria that can be cultivated from 100 ml
of water sample.
Streptococcus faecalis dies in water quickly, however its evaluation is important, since, as
opposed to E. coli (which can be found almost anywhere), S. faecalis does not reproduce in
the nonfecal environment. Thus its presence underlines the results of E. coli testing and
indicates the fresh fecal contamination of water. Anaerobic spore forming Clostridium welchii
stays viable in water for a long period of time, thus its presence in water indicates heavy and
long-lasting fecalcontamination of water.
A. THE TRANSFER OF THE UNIDENTIFIED STRAIN TO FRESH MEDIA (2 COPIES):
Strains:
Culture medium:
B. MICROBIOLOGICAL EXAMINATION OF SURFACE WATER; HYGIENIC CONTROL
B/1. COLI-COUNT DETERMINATION BY MEMBRANE FILTER TECHNIQUE
A membrane filter with a 0.45m pore-size is generally used to remove bacteria from
solutions.
Exercise: Filter 100 ml of a water sample through a 150m thick and 0.45m pore-sized
sterile membrane filter. Remove the membrane filter and place it facing up onto the surface of
52
differential media (endo- or eosin-methylene blue agar plates). Incubate the plates at 37C for
24 hours and count the number of colonies formed. Knowing the volume of water that has
been filtered, we can estimate the Coli-count of 100 ml water sample.
Sample:
water sample (river water, well water)
Materials:
graduated cylinder (to measure a 100 ml of water), 0,45m pore-size
membrane filter (for filtering tap water or natural water samples), sterile filtering unit, Endo
agar Petri plates (LES Endo Agar) or Eosin-Methylene Blue agar plates (EMB Agar), metal
forceps (use alcohol for flaming)
B/2. COLI-COUNT
DETERMINATION BY
MICROTITER PLATES
MPN
TECHNIQUE USING
LMX-BROTH
IN
96
WELL
The borderline dilution method is based on a serial dilution prepared form a sample and
inoculation of the special (giving a typical reaction for the given group of microorganisms)
differential media with the same amount of suspension of each dilution. Following the
predefined incubation conditions (time and temperature), the most probable bacterial count
(MPN most probable number) could be estimated from the number of tubes indicating
bacterial growth by mathematical/statistical calculations. This method presumes that the last
dilution, the last test tube where bacterial growth could be observed, contains only one cell.
The probable bacterial count of 1 ml of sample with this technique can be estimated.
During this practical, we employ LMX medium containing MUG substrate which can be
cleaved by the GUD enzyme, cleaving off a compound that gives fluorescence under 366 nm
wavelength UV light. Among the Gram-negative bacteria, 96% of E. coli, 100% of
entorotoxic E. coli, 44% of certain Shigella species and 17% of bacteria belonging to the
Salmonella genus produce the enzyme
53
Sample:
Materials:
water sample (river water, well water)

LMX broth, 50 ml Erlenmeyer flask, pipette and tips, UV-lamp of 366
nm wavelength, 96 well microtiter plates, test tubes, vortex, circular shaker
Exercise: Prepare a serial dilution from the water sample as follows: Measure 3g/ml water
sample into 27 ml LMX broth in 50 ml Erlenmeyer flask. Homogenize the sample at 400
RPM (rounds per minute), at 20C for 15 minutes on a circular shaker. Prepare an 8-member
dilution series from the obtained suspension so that you carry 0,3 ml of the sample suspension
into a test-tube containing 2,7 ml of LMX broth. Mix vigorously (vortex) then again transfer
0,3 ml suspension into a new test tube, and so on. From each dilution series (including the
original sample), pipette 300 l onto a 96-well microtiter plate in five parallels (5 test-tube
MPN method). Incubate the microtiter plates at 37C for 24 hours. Evaluation of the results:
the E. coli count is determined by counting the fluorescent wells (under UV-lamp of 366 nm
wavelength) of the plate and transforming it to the appropriate McCardy characteristic
number.
Table 10. 1. McCardy-table

B/3. EXAMINATION OF COLIFORMS ON ENDO- OR EOSIN-METHYLENE BLUE AGAR PLATES
Eosin-Methylene Blue agar is one of the most efficient differential media. It differentiates
lactose fermenting bacteria based on the fact that the acid produced by lactose fermentation
precipitates eosin, which is colored by the methylene blue dye content of the medium, so that
the bacterial colonies on the surface of the Eosin medium display a purplish blue coloration.
The medium also contains sucrose which helps the groth of other microbes but later the dye
inhibits growth.
Within the Endo-agar: the dark red alkaline fuchsin is decolorized by sodium sulfite
(Na2SO3). Acetaldehyde is one of the intermedier compounds during lactose fermentation,
54
which binds sulfite, thus the color of the fuchsin becomes visible. The typical coliform
organisms dye the Endo-agar red and grow on the surface of the agar as dark red colonies
having a metallic shine.
Materials: Endo agar Petri plates (LES Endo Agar) and Eosin-Methylene Blue agar plates
(EMB Agar), 9 ml sterile water containing test tubes (for the preparation of
dilution series), glass rod (alcohol for flaming), sterile pipet and tips
Exercise B/3: Prepare a serial dilution from the water sample and spread 0,1-0,1 ml of each
dilution onto the surface of Endo-agar and Eosin-Methylene Blue agar plates. Incubate the
plates at 37C for 24 hours and evaluate the number of typical coliform colonies.
B/4. CULTURING PSEUDOMONADS ON BROLACINE AGAR
Member of the Pseudomonas family are widely distributed in soil and water. The
Pseudomonads are Gram-negative, strictly aerobic, mostly obligate aerobic bacteria having
polar flagella. Among them, Pseudomonas aeruginosa is a common bacterium of different
waters, wastewater and contaminated water. For the demonstration of the members of the
genus Pseudomonas, brolacin nutrient medium is used, which contains lactose, peptone,
cystine and brom thymol blue indicator (green at neutral pH). The colonies of Pseudomonads
appear brown in the centre and dye the surrounding area of the agar blue. The presence of
Pseudomonads does not indicate fresh contamination, however it can often be found inside
wet hospital units (taps, pipelines,etc.) and equipment. This microbe usually attacks patients
who are weak, immune suppressive and received radiation or antibiotic treatment.
Materials:
Brolacin agar, glass rod (alcohol for flaming), sterile pipet and tips
Exercise: Spread 0,1-0,1 ml of each dilution onto the surface of Brolacin agar plates. Incubate
at 37C for 24 hours, observe the brown centered colonies with blue zones around them.
Calculate a Pseudomonas-count from the amount of such colonies.
B/5.DEMONSTRATION OF STREPTOCOCCUS OF FECAL ORIGIN USING SZITA-E-67 MEDIUM
Members of the Gram-positive coccus the genus Streptococcus produce shorter-longer chains.
Some of them are human and animal pathogens but healthy human or animal organisms can
also carry these bacteria. In clinical, epidemiological laboratory practice, the Szita E-67 agar
medium is generally used for the cultivation of Streptococcus. The selectivity of the Szita E67 medium for Streptococcus is assured by the mediums tellurite and Na-taurocolate content.
Streptococcus faecalis reduces the tellurite within the medium and forms black colonies on
the surface of the Szita E-67 agar, while other bacteria hardly growth at all.
Materials:
Szita-E-67 agar, glass rod (alcohol for flaming), sterile pipette and tips
Exercise: Spread 0,1-0,1 ml of each dilution onto the surface of Szita-E-67 agar plates.
Incubate at 37C for 24 hours, observe the characteristic black Streptococcus colonies.
Calculate a Streptococcus-count from the amount of such colonies.
55
B/6. DETERMINATION
OF COLIPHAGES FROM SURFACE WATERS BY THE POUR-PLATE-
TECHNIQUE
Bacteriophage (viruses that infect bacteria), often called phage, are by far the most
convenient viruses for laboratory investigations. Phages are rapidly replicated in their
bacterial hosts. The phage that infects Escherichia coli is called coliphage. Coliphage is added
to the bacteria in soft agar prior to pouring the plate, iff the coliphage lyse (burst) cells while
Escherichia coli is multiplying in the overlay, plaques (clear areas) develop in the lawn of
bacteria. The culturing of the bacteriophage of the fecal contamination indicating E. coli from
an environmental sample is a new technique in the microbiological analysis of water samples.
Exercise: Measure 100 ml water sample into a sterile flask and place it into a water bath of
45C-47C. Place 4 ml sterile CaCl2 solution into the same water bath for 10 minutes.
Meanwhile, dissolve 0,05g of TTC (triphenyl-tetrazolium-chloride) in 5 ml 96% ethanol.
Then measure 1 ml of the 1% TTC solution and 1,2 ml of the CaCl2 solution into 100 ml
liquefied nutrient agar. Inoculate the water sample with 5 ml of a 24 h Escherichia coli
(ATTC 13706) suspension previously shaken at 37C. Carfully and slowly pour the agar into
the water sample and slowly mix it, then pour it out into a big Petri dish avoiding the
formation of any bubbles. The viable bacterial cells reduce TTC, which turns the agar red,
while white plaques form where the phage has destroyed the bacterial cells.
C. TSI MULTITEST MEDIUM FOR THE DIFFERENTIATION OF GRAM
NEGATIVE BACTERIA
Because of their complexity, multitest media can be used for testing several enzymatic
activities simultaneously. TSI (Triple Sugar Iron) agar is generally used for the
characterization of enterobacteria. The medium is rich in protein and contains 0,1% glucose,
1,0% sucrose and lactose, ferrous sulfate and phenol red indicator. Phenol red changes to
yellow when the environment is acidic.
The intestinal pathogens Salmonella and Shigella ferment dextrose but not sucrose or lactose.
The most common Gram negative, non-pathogenic fecal rods do not share these
characteristics.
Strains:
Materials:
Enterobacteria
TSI agar slants, inoculating loop
Exercise: Inoculate the TSI agar slants with a stab inoculation as well as a zigzag strak
inoculation on the surface of the agar slant. Place the tubes into the 28C thermostat for 2-7
days. Evaluate the observed features of the agar as follows:
Evaluation:
1. Yellow stab inoculation and red slant acid production from glucose degradation
(because only the fermentation of a small amount of glucose (0.1%) present in the
medium happens, only the bottom, anaerobic zone becomes acidic.)
2. Yellow stab inoculation and yellow slant acid production from lactose and/or
sucrose degradation (because the fermentation of large amounts of sucrose and/or
lactose (1%) happens, the whole nutrient medium becomes acidic and yellow.)
3. Formation of bubbles or cracks intensive fermentation, gas formation
4. Red stab inoculation and red slant nor carbohydrate degradation or gas formation
5. Black coloration indicates H2S production
56
TSI multitest media

Enterobacter
Escherichia
Klebsiella
Proteus vulgaris
Serratia
Shigella
Salmonella typhi
Stab
inoculation
yellow
yellow
yellow
yellow
yellow
yellow
yellow
Slant surface
inoculation
yellow
yellow
yellow
Yellow or red
Red or yellow
red
red
H2S production
Gas production
+/
Typical TSI reaction observed with some enterobacteria

D. EVALUATION OF THE EFFECT OF ANTIMICROBIAL AGENTS
57
PRACTICAL XI
A. THE MAIN PROCESSES INVOLVED IN THE NITROGEN CYCLE ARE:
Assimilative nitrate reduction
Organic-N
Ammonification
and
NH3
assimilation
N-fixation
NH3
Dissimilative nitrate reduction
N2O
N2
NO2-
Nitrification
NO3-
Nitrification
The oxidation number of Nitrogen
-3
+1
+3
+5
FIG. 11.1. The nitrogen cycle

A/1. NITROGEN FIXATION: THE EXAMINATION OF NITROGEN FIXING MICROORGANISMS
Biological nitrogen fixation is carried out by prokaryotic organisms. During the process of
nitrogen fixation, the oxidation state of nitrogen atoms is reduced from 0 (nitrogen gas) to -3
(ammonia). The ammonia gained by nitrogen fixation could later be assimilated into organic
compounds. The structure of the nitrogenase enzyme-system of the various nitrogen-fixing
organisms differ only slightly, thus these organisms fix nitrogen practically through similar
reaction.
However, these nitrogen-fixing organisms can be divided into two major groups: free-living
organisms (e.g. Azotobacter, Clostridium, Anabena) and symbionts (close association). The
latter group consists mostly of the members of the genus Rhizobium, which is associated with
leguminous plants (e.g. peas, beans, soybean). Free-living rods infect the root hairs of
leguminous plants. The plant responds by producing nodules to wall off the infection. Inside
these swellings, Rhizobium cells grow, becoming pleomorphic symbionts called bacteroids.
Bacteroids have characteristic morphology and they fix atmospheric nitrogen.
Among the nitrogen-fixing prokaryotes, aerobic, microaerophile and anaerobic
microorganisms can be identified. Since the nitrogenase enzyme is very sensitive to the
presence of oxygen, thus in an aerobic/oxic environment the intracellular oxygen tension
should be reduced. The nodules contain leg-hemoglobin, which is responsible for the suitably
low oxygen tension needed for the bacteroids.
Cyanobacteria - morphology
Cyanobacteria, also known as blue-green algae, blue-green bacteria, is a phylum of
bacteria that obtain their energy through photosynthesis. The name "cyanobacteria" comes
from the color of the bacteria. They are a significant component of the marine nitrogen cycle
and an important primary producer in many areas of the surface waters Cyanobacteria were
58
traditionally classified by morphology into five sections, referred to by the numerals I-V. The
first three - Chroococcales, Pleurocapsales, and Oscillatoriales - are not supported by
phylogenetic studies. However, the latter two - Nostocales and Stigonematales - are
monophyletic, and make up the heterocystous cyanobacteria. The members of Chroococales
are unicellular and usually aggregate in colonies. The classic taxonomic criterion has been the
cell morphology and the plane of cell division. In Pleurocapsales, the cells have the ability to
form internal spores (baeocytes). The rest of the sections include filamentous species. In
Oscillatoriales, the cells are uniseriately arranged and do not form specialized cells (akinetes
and heterocysts). In Nostocales and Stigonematales, the cells have the ability to develop
heterocysts in certain conditions. Stigonematales, unlike Nostocales include species with truly
branched trichome.
Exercise: Study of water samples for Cyanobacteria; study of heterocysts
Heterocyst: Heterocysts are specialized nitrogen-fixing cells formed by some filamentous
cyanobacteria, such as Nostoc punctiforme, Cylindrospermum stagnale and Anabaena
sperica, during nitrogen starvation. They fix nitrogen from dinitrogen (N2) in the air using the
nitrogenase enzyme in order to provide the cells in the filament with nitrogen for biosynthesis.
Nitrogenase is inactivated by oxygen, so the heterocyst must create a microanaerobic
environment. The heterocysts' unique structure and physiology requires a global change in
gene expression. For example, heterocysts:
Examination of cyanobacterial morphology
Fig. 11.2. Morphology of cyanobacetria

59
A.2. Examination of bacteroid morphology

Sample: root sample (leguminous plant)
Materials: microscope slide, methylene-blue dye, scalpel and forceps
Exercise: Cut a nodule off the washed leguminous root sample using forceps and a scalpel.
The active nodules have a slightly pink color, since they contain leg-hemoglobin. Gently press
the nodules between two microscope slides. Air dry and heat fix the smear, then stain the
smear with methylene-blue. Observe the large, irregular rods of the bacteroids under the
microscope.
B. DEMONSTRATION OF AMMONIFICATION
During ammonification, some nitrogenous compounds (amino-acids, carbamide etc.) are
transformed into ammonium ions and ammonia is freed. For such deamination reactions,
bacteria utilize many enzymes with different substrate specificity (one way of getting rid of
excess organic nitrogen). The remaining part of the molecule can be used for energy
generation . Within the nitrogen cycle, ammonification is considered as a mineralization step
from which ammonia and ammonium ion can be taken up and used for amino-acid synthesis
by other organisms (plants, microbes) or it can be absorbed in the soil by humus-colloids.
NH3
Potassium-mercury(II)-iodide
Mercury(II) [amido-triiodide-mercurate(II)]
orange complex
Sample: own unidentified strain

Materials: peptone broth, urea broth, sterile water in test tubes, Nessler-reagent
Exercise: Inoculate the peptone broth containing test tubes with 0,1-0,1 ml bacterial
suspension and incubate the tubes at 28C for a week. The presence of accumulated ammonia
in the peptone broth can be demonstrated by adding a few drops of Nessler-reagent (an
alkaline solution of Potassium-mercury(II) iodide). Weak positive reaction yields a yellow
color, strong positive reaction results in a yellowish brown color and precipitation.
C. DEMONSTRATION
(ALLYL-THYOUREA)
OF NITRIFICATION AND INHIBITION OF NITRIFICATION WITH
ATU
During aerobic conditions, ammonia does not accumulate in soil or water. Beside the
assimilation of ammonia (for amino-acid synthesis), certain bacteria can gain energy by
utilizing ammonia as electron donor (as well as producing reducing potencial) in dissimilative
processes. Nitrification is carried out by chemolitho-autotrophic bacteria (e.g. Nitrosomonas:
NH3NO2- and Nitrobacter NO2-NO3-) and different heterotrophic microorganisms. Nitrite
can be taken up by plants more easily than ammonia; however, because of its bigger mobility,
nitrite can be leached from soil, deteriorating the quality of both surface and underground
waters.
Nitrification is a two-step process both chemically and biologically:
60
Name of process
Reaction
Typical bacterial genus
Ammonia oxidation
NH4+ + 1,5 O2 NO2- + H2O + 2 H+
Nitrosomonas, Nitrosospira
Nitrite oxidation
NO2- + 0,5 O2 NO3-
Nitrobacter, Nitrospira
The key enzyme in ammonia-oxidation is ammonia mono-oxigenase (Amo). The enzyme is

active in the presence of copper ions. Allyl-thiourea (ATU) is a selective copper-chelator
compound, thus ATU can inhibit the ammonia mono-oxigenase enzyme.
Sample: soil suspension, own unidentified strain
Materials: Ammonia and nitrite containing broth, Nessler-reagent, Griess-Ilosvay-reagent
(nitrite reagent), zinc powder, ATU
Exercise: Add ATU to half of the nutrient media, so that the final concentration would be 5
mg/l. Inoculate the media with 100 ml soil suspension. Incubate the samples at 28C for a
week.
Evaluation of results: Transfer 1-1 ml of each broth to a new, empty test tube. Add some
drops of Griess-Ilosvay-reagent to the tubes (nitrite A and B reagent). Mix the contents of the
tube. The presence of nitrite in the broth is demonstrated by a cherry red color within 30 sec.
There are two possibilities if there isnt red coloration: either ammonia oxidation has not
taken place (effect of the inhibitor), or nitrite was completely oxidized to nitrate. In order to
be able to distinguish between these two options, add a small amount of zinc to the tubes
(max. 5 mg/ml). If nitrate has formed during nitrification, zinc would reduce it back to nitrite,
and the Griess-Ilosvay-reagent will form a red coloration. If there is still no color change after
the addition of zinc, add Nessler-reagent to the remaining broth. If a yellowish brown color
develops it indicates the unchanged ammonium ions.
If there is no red coloration in the nitrite-containing broth, then nitrite oxidation was
complete. To prove this assumption, perform the zinc reduction test.
Nutrient media
The presence of which compound What kind of oxidation has

have you demonstrated?
taken place?
+
Ammonia
Nitrite
NH4
NO2
NO3
Ammonia
Ammonia + ATU
Nitrite
Nitrite+ ATU
Fig.11.3. Evaluation of experiments used for the demonstration of nitrification
D. DEMONSTRATION OF DISSIMILATIVE NITRATE REDUCTION
During the dissimilative reduction of nitrate or nitrite, the end products are nitrite,
dinitrogen-oxide, nitrogen gas or ammonia. The process is anaerobic and takes place in
compacted, water saturated soils; river or lake sediments; inside the gastrointestinal tract of
higher organisms, etc. When a gaseous substance (N2O, NO, N2) is produced, the process is
called denitrification. This process plays a very important role in the nitrogen equilibrium
and self purification of soil and water. From the biochemical point of view, this process is
61
nitrate reduction: the utilization of nitrate as electron acceptor for the biological oxidation of
different organic and inorganic (e.g. H2S) substances.
Sample:
Materials:
own unidentified strain, soil suspension

Nitrate broth containing Durham test tubes, zinc powder, Nesslerreagent, Griess-Ilosvay-reagent (nitrite reagent)
Exercise: Inoculate the nitrate media with 1 ml soil suspension. Incubate the samples at 28C
for a week and evaluate the results for the presence of the following products:
Nitrite (NO2-):
Griess-Ilosvay reagent
Nitrogen gas (N2):
Bubbles inside the Durham tubes
Ammonia:
Nessler-reagent
E. EVALUATION OF RESULTS OF WATER ANALYSIS
62
PRACTICAL XII
FERMENTATION PROCESSES IN BIOTECHNOLOGY
Biotechnology in the classical sense is defined as an industrial production technique, where
some living organism or components of them (e.g. enzymes) carry out the production of the
required product. Technologies based on microbial fermentations can be defined as
traditional biotechnological techniques. Through in microbial fermentations, microorganisms
in an artificial environment in fermentors (at aerobic or anaerobic conditions) produce
materials (primary or secondary metabolites), such as antibiotics, citric acid, alcohol.
Nowadays the use of recombinant DNA techniques made it possible to produce several
compounds (e.g. insuline, growth factors, interferon etc.) by genetically modified living
organisms. (Table 12.1). These organisms can be plants and animals or their cell cultures
besides microorganisms.
Metabolites
Microorganisms
Products intended for industrial use
Ethanol (from glucose)

Ethanol (from lactose)
Acetone and butanol
2,3-butanediol
Enzymes (amylase, protease etc.)
Gibberellins
Saccharomyces cerevisiae
Kluyveromyces fragilis
Clostridium acetobutylicum
Enterobacter, Serratia
Aspergillus. Bacillus, Mucor, Trichoderma
Products intended for agricultural use

Gibberella fujikuori
Food additives
Amino acids (lysine etc.)

Organic acids (citric acid)
Nucleotides
Vitamins
Polysaccharides
Corynebacterium glutamicum
Aspergillus niger
Corynebacterium glutamicum
Ashbya, Eremothecium, Blakeslea
Xanthomonas
Products intended for medical use

Antibiotics
Alkaloids
Steroids
Insulin, human growth factor,
interferon, somatostatin, etc.
Penicillium, Streptomyces, Bacillus

Claviceps purpurea
Rhizopus, Arthrobacter
Escherichia coli, Saccharomyces cerevisiae
and other organisms (recombinant DNA
techniques)
Products used as fuels
Hidrogen
Methane
Ethanol
Photosynthetic microorganisms
Methanobacterium
Zymomonas, Thermoanaerobacter
63
Microbial metabolites can be divided into two groups: primary and secondary metabolites.
The primary metabolites essential for cellular life and reproduction are synthesized during the
growth phase (trophophase). These substances include: amino acids, nucleotides, fermentation
end products (e.g. ethanol and organic acids). The secondary metabolites however are
produced in the idiophase, following the growth phase. The cell no longer replicates and
grows through changes that can be accounted for lack of nutrients, accumulation of toxic
substances, lack of oxygen or other essential compounds needed for the synthesis of cell
components, growth and replication. These substances accumulate within the nutrient
medium, they differ in their chemical structure and physiological impact (e.g. antibiotics,
micotoxins).
A.1. CITRIC ACID PRODUCTION IN SHAKEN CULTURE
Nowadays,
citric
acid
(2-hydroxil-propane-1,2,3-tricarbolicacid)
is
produced
microbiologically by Aspergillus niger and Aspergillus wentii. The most important carbon
source for citric acid production is glucose. Some of the glucose is used up during culturing
for mycelium growth in the trophophase, while glucose is used for citric acid production
during the idiophase.
Exercise: Take Aspergillus niger spores from an agar slant into sterile distilled water using an
inoculating loop. Set spore concentrations of the suspensions at 20,000 and 80,000 spores/ml
using the Brker chamber. Add 1 ml of each spore suspension into separate Erlenmeyer flasks
containing 50 ml nutrient media for citric acid production. Shake the flasks at 37C for a
week at 270 rpm. Demonstrate the citric acid production by thin layer chromatoghraphy
(TLC).
Strain:
Materials:
Aspergillus niger
Brker chamber, thin layer chromatographic sheets, microcapillaries,
forceps, hair dryer, nutrient media for citric acid production
Fig. 12. 1. Brker chamber

Immobilised cell culture fermentation of alcohol
Alcohol production, in the industry, by Saccharomyces cerevisiae cells. Advantages of
immobilized cell technique are:
1. Higher cell density
2. microbial biomass can be used several times
3. continuous fermentation
4. less possibility of contamination
5. product can be easily purified
64
Techniques:
Binding to a carrier
Covalent bond
adsorption
Capturing
Cross binding
=cell or enzyme
Fig. 12. 2. Immobilized cell techniques

Binding to a carrier is based on the technique that the cells or enzymes are bound to a solid
carrier with ionic or covalent bonds. Carriers can be not water soluble polysaccharides
(cellulose, dextrane, agaroise), proteins (gelatine, albumine), synthetic polymers (ion
exchange resins, polyvinyl chloride) and inorganic compounds (quartz). At the cross-binding
method, 2 or more function group reagents (glutarealdehide) will react with the cells. At the
capturing method, cells will bind into a polymer material (alginate, polyacrylamide).
Exercise:
Saccharomyces cerevisiae cells will be immobilized in Ca-alginate gel and will ferment
alcohol from glucose and malate.
50 ml Saccharomyces cerevisiae cell suspension (about 25 g wet cell biomass) sterily mixed
with 4% alginate solution, then this will be dropped into 0.15M CaCl2 (pH6-8) solution at
37C. Solidify for 1 hr at 20-22C, then stabilize overnight at 4C. Solidifying solution is
changed to glucose solution next morning, fermentation at 28C.
Next week, alcohol is detected with H2SO4 diluted potassium/dichromate.
Strain: Saccharomyces cerevisiae
65

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MICROBIOLOGY

PRACTICAL GUIDE (A)

Fig. 1.1. Cascade impactor

Water (1-2 cm)

Non-sulfur photosynthetisers (rust red)

"Purple" zone (anaerobic sulfur bacteria)

2kgs shredded cabbage

H-donors and acceptors (approximately 1-15 g/L)

In routine bacteriology laboratory exercises, complex or nonsynthetic media are

CFU/mL or g: number of colonies on plate x 1/dilution rate

Fig. 2.2. Usage of spreading rod

D. Evaluation of the results of enrichment technique.

Fig. 3.1. Basic colony morphology types

It is important to keep a Bunsen-burner on throughout the process to prevent contamination

Fig 3. 2. The proper way of sterilizing the inoculating loop

D. Preparation of pure cultures

Fig. 3.4. Preparation of pure cultures

solved by using Marino-plates (better known as Brewer-plates), a combination of a Petri-dish

Figure 4.1 The anaerobic jar system

and in wastewater treatment processes (part of the biofilm-forming community). Therefore it

pH: 7,0-7,5, sterilization: 1 atm, 121C, 15 min

B/5. Demonstration of the anaerobic chamber (glove-box)

Own unidentified strain

B/2. Temperature tolerance

C. Effect of pH on microbial growth

D. The effect of UV radiation on bacterial growth

Own unindentified strain

E.Osmotolerance of microorganisms, effect of water activity (aw) on microbial growth

Water Activity of Various NaCl Solutions

Water Activity (aw)

Water Activity (aw)

F. Demonstration of the efficacy of different disinfectants

G. Microbes digesting pectine

G/2. Soft-rot test

The components of the microscope

1. ocular lens, or eyepiece 2.

All optical microscopes share the same basic components:

How a microscope works

Limitations of light microscopes

CURVED ROD (SPIRAL SHAPE)

Actinomyces have branching cells, forming Streptomyces

Table 6.1. Morphology of bacteria

B.1. SIMPLE STAINING

Color changes due to the different steps of Gram staining

Gram positive cell

Gram negative cell

Fig. 6.1. Motility of bacteria studied by indirect method

D. Checking the pH of the sour-cabbage

Gram staining from supernatant of the cabbage

Make smear and fix it

Heat + Carbol Fuchsin

Fig. 7.1. Acid fast staining

Staining: Own bacterial strain, Rhodococcus rhodocrous; Staphylococcus aureus

Prepare smear, fix with heat

Exercise: Bacillus cereus; Saccharomyces cerevisiae; own bacterium

B/1 CATALASE ACTIVITY

B/3. METHYLENE BLUE REDUCTION

Own unidentified strain (24h on agar slant), Escherichia coli

C/1. CASEASE ACTIVITY (casein hydrolysis)

Own unidentified strain (24h on agar slant), M. luteus, B. subtilis

Own unidentified strain (24h on agar slant)

Own unidentified strain (24h on agar slant)

(Tween 80 is the oleic acid ester of a polyoxyalkylene derivative of sorbitan)

Own unidentified strain (24h on agar slant)