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BASC 5205
Tri II
By:
Anjum Odhwani, MD MPH
1
TABLE OF CONTENTS
Microbiology Laboratory Instructions .
Supply List ..
Hand Washing .
Transmission of Disease ..
Microscopy ..
Aseptic Technique
Gram Stain
Endospore Stain.
Motility
Anaerobes .
Microbiological Media..
The Staphylococci
The Streptococci ..
The Neisseria ..
The Enteric .
Preparation of yogurt
SAFETY: Living organisms will be used. Some cause disease while others do not. However,
any organism will cause disease under certain circumstances. Therefore, be sure to follow all the
instructions. Safety for the whole class depends on you.
GENERAL INSTRUCTIONS:
1. BRING only your lab manual, markers, colored pencils and lab coat to lab. LEAVE
personal belongings outside of the lab. There is no place to keep them in the lab.
2. WASH the table top with disinfectant before and after lab. (Antiseptic________,
Disinfectant__________)
PERSONAL ITEMS:
1. NO smoking, eating, drinking or gum chewing in the lab.
2. DO NOT touch fingers to your face, ears, eyes, etc. KEEP pencils out of your mouth.
4. REPORT any open cuts or scratches to the instructor before working on an experiment.
USE antiseptic and bandage wounds. WASH hands well after the lab.
Staining Tray
Clothes Pin
Lens Paper
Inoculation loop and needle
Bibulous Paper
Forceps
Ruler Clothespin
Microscope Slides
Bunsen burner
Lens paper
Striker/Lighter
Bibulous paper
Bunsen Burner
Striker
Forceps
Ruler
Hand Washing
Objectives
Describe and demonstrate proper hand washing technique;
Describe the importance of proper hand washing technique in prevention of transmission of
pathogens.
Material
1. Sink
2. Paper Towel
3. Water
4. Soap
Procedure
1. Wet your hands with clean, running water (warm or cold), turn off the tap, and apply soap.
2. Lather your hands by rubbing them together with the soap. Be sure to lather the backs of
your hands, between your fingers, and under your nails.
3. Scrub your hands for at least 20 seconds.
4. Rinse your hands well under clean, running water.
5. Dry your hands using a clean towel or air dry them.
6. Turn off the water using the same paper towel to avoid recontamination of your clean
hands.
2. Disease - any adverse internal condition severe enough to interfere with body functioning.
6. Vectors Any living organism that can carry pathogens from one host to another. The
most common vectors are the arthropods such as insects eg. Mosquito, fleas, lice
and ticks.
a. Biological vector Organism that transmits a pathogen and within which the
pathogen can multiply to high numbers.
b. Mechanical vector Organism such as fly that physically moves contaminated
material from one location to another.
7. Direct Transmission - pathogens are passed from one person to another by direct body
contact examples are
a. Skin to skin contact eg. Herpes labialis or cold sore
b. Sexually transmitted eg. Genital herpes, HIV, Gonorrhea etc.
c. Transplacental Transmission eg. Rubella, Toxoplasmosis and Syphilis
transmitted from mother to fetus.
8. Indirect Contact Spread of pathogens from one host to another via any of the following
methods.
a. Fomite Transmission- inanimate objects that transmit disease such as spoons,
cups, bedding, etc.
b. Droplet Transmission Spread of pathogens from one host to another via
respiratory droplets. People in close proximity can inhale those droplets, resulting
in disease. In droplet transmission a cloud of water droplets travel less than 1
meter.
c. Aerosol Transmission A cloud of water droplet can travel more than one
meter.
d. Fecal-oral transmission - usually from contaminated food or water sources
10. Antiseptic- Chemical used to inhibit or kill microorganisms on living cells and tissues.
12. Sterilization- The process of destroying or removing all microorganisms and viruses
through physical or chemical means.
14. Pasteurization- Use of heat to kill pathogens and reduce the number of spoilage
microorganisms in food and beverages.
OBJECTIVES:
1. To observe how pathogens can be transmitted by touching.
2. To observe how transmission can be prevented.
3. To observe which method of washing hands is most effective in preventing disease
transmission.
MATERIALS:
1. Yeast suspension Fleishmans commercial yeast for baking (Brewers yeast), it is a
member of the genus Saccharomyces. Brewers yeast is considered non-pathogenic.
Please still use caution when handling this organism. If there is a possibility of an
allergic reaction please do not directly participate in this experiment but act as an
observer.
2. Sabourauds Dextrose agar has a relatively high sugar concentration (40g/L) and a low
pH of 5.6; therefore these conditions favor fungi over bacteria.
3. Hand sanitizer
4. Soap and water
QUESTIONS
2. In your opinion, which is the most effective method to prevent disease transmission?
DEFINITIONS:
1. Septic: characterized by the presence of pathogenic microbes in living tissue.
5. Agar: a seaweed polysaccharide containing D and L galactose; agar makes broth solid.
Microorganisms cannot digest agar, therefore, the medium remains hard.
7. Petri Plates: flat plastic plates with a lid; usually filled with a solid medium.
DEMONSTRATION:
OBJECTIVES:
Demonstrate aseptic inoculation of broth medium.
Describe growth characteristic in broth
Define pure culture and a bacterial colony
Explain the purpose of a four quadrant streak plate.
Demonstrate how to make a four quadrant streak plate.
Describe the characteristics of an isolated colony (form, margin and elevation)
Bacteria have growth characteristics in broth cultures. After inoculation of broth media, bacteria
may exhibit a particular form of growth. These include the formation of a thin surface film called
pellicle (obligate aerobe), accumulation of the cells at the bottom called sediment (obligate
anaerobe and facultative anaerobe), and clouding of the medium called turbidity (facultative
anaerobe).
Bacteria are found in mixed cultures in many environments like food, soil, water, and the human
body. A pure culture must be obtained before the characteristics of the organism can be studied.
A pure culture is a culture which has a single type of organism. In this exercise you will learn
the most common technique for isolating organisms from each other. The purpose of four
quadrant streak plate is to isolate a bacterial colony. In the streak plate method, the mixture is
thinned out on an agar plate so that individual organisms are separated from each other. Each
organism forms a colony. A colony is an aggregation of cells arising from a single parent cell. A
colony of organism that comes from a single organism is a pure culture because all the organisms
will be genetically identical. In a colony there are one million bacteria. The pure colony can be
further cultured and characterized.
The characteristics of a bacterial colony are determined by the genetic makeup of the cell.
Bacterial colonies have a particular
Form (shape)
Margin (edge)
Elevation (Raised, convex, umbonate)
Size (punctiform, small, moderate and large)
Appearance glistening or dull
Pigmentation nonpigmented (cream, tan, white) pigmented (purple, red, yellow)
Optical characteristic (opaque, translucent, transparent) of the colony depending on the
bacterial type.
You will observe the colony types produced by the bacteria found in a mixture.
MATERIALS:
1. nutrient agar plates (TSA) - one per person
2. nutrient broth tubes (TSB) or (BHI) - 4 tubes one per person
3. inoculating loop
4. pure bacterial cultures - Serratia, B. subtilis, E. coli, Staph. epidermidis.
5. a mixture of organisms - (Serratia, E. coli, Staph. epidermidis)
Quadrant 1 - Apply a
loop full of culture Quadrant 2
Flame to
start
Flame
loop
1
2
Flame loop
Quadrant 3
Quadrant 4
Flaming 4
optional
370 C
B. subtilis pellicle
STREAK PLATE
370 C
E. coli F - irregular
M - undulate
E - umbonate
C - cream
Serratia F - circular
M - entire
E - umbonate
C - red
B. subtilis F - irregular
M - Lobate
E - flat
C - cream
Mix culture
5. What is the name of solidifying agent used most successfully in bacterial nutrient
media?
Broth Characteristics
Staphylococcus epidermidis
Bacillus subtilis
Serratia marcescens
E. coli
Staphylococcus
epidermidis
Bacillus subtilis
Serratia marcescens
E. coli
OBJECTIVES:
1. Explain the difference between a simple and compound microscope and be able to name
some of the other types of scopes currently in use today.
2. Identify various parts of a compound microscope including the: condenser, diaphragm,
course and fine focus adjustments, objectives, revolving nosepiece and rheostat.
3. Define resolving power, total magnification, parfocal, working distance, real image, and
virtual image.
4. Demonstrate how to use and clean a microscope.
Historically, Anton van Leeuwenhoek is credited with initiating the use of the microscope as a
research tool. Using a simple microscope, he described many bacteria and protozoa in 1674.
The single lens, simple microscope was impractical due to the limitations in magnification and
resolution. The compound microscope with a double lens system was developed over the next
two centuries. The compound microscope that we know today, with a sub-stage condenser and
oil immersion lens, was designed by 1866. Refinements since then have made the light
microscope the powerful tool it is for the observation of microorganisms. The optics are
excellent, and the microscopes are almost mechanically flawless. The phase contrast, the
fluorescence and the electron microscopes have been developed since World War II.
The microscope is a quality tool, but it is useless without knowledge of how to achieve the
maximum resolution, the greatest magnification and other tricks of the trade. The resolution or
the sharpness of the image depends on the adjustment of the diaphragm, condenser and the use of
immersion oil. Haphazard adjustments will cause distortions in the image. This lab is designed
to familiarize you with the strengths and weaknesses of your microscope. A definition sheet has
been prepared to help you understand this instrument.
Questions
1. How is the total magnification of a microscope determined?
OBJECTIVES:
Some of the criteria for classifying or identifying bacteria are the shape, size and arrangement of
the bacteria. These characteristics can be visualized in the microscope when the organisms are
applied to a slide and are stained. The three basic shapes are cocci, bacilli and spirals. This
exercise will demonstrate the application of bacteria to a slide, the simple stain procedure and the
handling of pure bacterial cultures.
The cultures you use will be pure cultures which contain a single type of organism. The cells
may be grown in liquid nutrient broth or on a solid nutrient agar. The agar used will be in slants
because the bacteria are easier to pick up from a slanted surface than from the surface of a deep.
The bacteria will be transferred to glass slides by aseptic (sterile) technique. This technique
insures that the cultures remain pure and that the environment is not contaminated. The bacteria
must be fixed to the slide so that the bacteria will not wash off during the staining procedure.
A bacterial smear is the fixed bacteria on a slide that are ready for staining.
When a simple stain is made, a single dye is used. Various dyes can be used. The only
requirement is that the dye must be a basic dye with a positive (+) charge, so that it will be
attracted to the negatively charged cytoplasm of the bacterial cell. Crystal violet, methylene
blue, safranin, and malachite green are positively charged basic dyes.
Staphylococcus epidermidis
Bacillus subtilis
Make Drawings
OBJECTIVES:
Hans Christian Gram developed a method of staining bacteria in 1874. He found that some
bacteria would retain their color after treatment with alcohol while others would lose the color.
The microorganisms could be divided up into two groups based on the results of this staining
procedure. The organisms that retain crystal violet stain are called Gram positive. Those that
lose crystal violet stain and retain safranin are called Gram negative. The (+) and (-) do not refer
to the electrical charge; they simply refer to the response of the organism to the staining
procedure. The Gram stain is still the single most important procedure in Microbiology because it
is the first biochemical step in the identification of organisms after an isolated colony is obtained.
Biochemically, the Gram stain works because of differences in the cell walls of bacteria.
Gram positive organisms have multiple layers of peptidoglycan in the cell wall.
Primary Stain
Crystal Violet is used first and stains all cells purple.
Mordent
Grams Iodine serves as a mordent, a substance that increases the cells affinity for a stain. It
binds to primary stain, thus forming an insoluble complex. The resultant crystal-violet-iodine
complex serves to intensify the color of the stain.
Decolorizing Agent
Acetone- Alcohol cannot easily remove the dye from the peptidoglycan. Its action is determined
by the concentration of lipids and the thickness of the peptidoglycan layer in bacterial cell walls.
In Gram negative cells the alcohol increases the porosity of the cell wall by dissolving the lipids
in the outer layers. Thus crystal violet iodine complex is easily removed from the thin layer of
peptidoglycan. The thick layer of peptidoglycan in gram positive bacteria will retain crystal violet
iodine complex.
Counterstain
Safranin is the final stain used to stain cells that have been decolorized. Since only Gram-negative
cells undergo decolonization, they will now absorb counterstain and will be stained red or pink.
Gram Stain
Make Drawing
6. Based upon the result of Gram Stain, bacteria are divided into how many groups?
7. Describe the cell wall of Gram positive and Gram negative bacteria?
a. Gram positive b. Gram negative
8. Under the microscope, Gram positive bacteria are of which color and gram negative
bacteria are of which color?
Endospores are heat resistant dormant form of bacteria. The genus Bacillus and Clostridium are
groups of organisms that produce endospores. The endospore is made up of many layers
including the spore coat and outer layer spore crust. It is the spore crust which protects the
bacteria from heat, drying and chemical agents including some antibiotics.
The endospore coat and core are stabilized with dipicolinic acid. Some of the diseases that are
caused by endospore-forming bacteria are Clostridium tetani causes tetanus, Clostridium
botulinum causes botulism, Clostridium perfringens causes gas gangrene and Bacillus anthracis
causes anthrax. Endospores can be visualized using the endospore stain.
Endospore Stain
MATERIALS:
Endospore Stain
1. Endospore producing organism - (Bacillus subtilis)
2. malachite green
3. safranin
4. steaming apparatus (Bunsen burner, ring stand, beaker of water and a wire gauze)
5. slides
6. bibulous paper
Endospore Stain
Sketch your results here
Endospore Stain
3. What is the name of the chemical present in the spore- coat and core that stabilizes
endospores?
OBJECTIVES:
1. Identify the biochemical difference between the cell wall of genus Mycobacterium and all
other bacteria
2. List the dyes used in Acid-fast stain
3. Demonstrate the steps of Acid-fast stain.
4. To recognize a Mycobacterium species from any other genus of bacteria.
5. Discuss the importance of an Acid-fast stain as a diagnostic test.
Some microorganisms particularly the members of the genus Mycobacterium has cell wall with a
high mycolic acid content and is visualized much better by acid-fast stain. Mycolic acid is a
group of branched chain hydroxy lipids. Mycolic acid prevents the cells from staining easily
with simple stains. Heating the specimen with carbolfuchsin (primary stain) and phenol allows
this red dye to penetrate the mycolic acid. Once the dye has penetrated the cell wall it cannot be
easily removed even with acidified alcohol. Hence, the organisms that stain with carbolfuchsin
that cannot be removed by acid-alcohol are said to be acid fast. Non-acid fast organisms have
the primary stain removed with acid-alcohol and can be counterstained with methylene blue.
Acid fast organisms are red while non-acid fast organisms are blue. This method is the Ziehl-
Neelsen method for the acid fast stain. Acid-fast stain is a differential stain.
Mycobacterium tuberculosis causes tuberculosis and M. leprae causes leprosy, acid-fast stain is
used as a diagnostic procedure for tuberculosis and leprosy.
Acid-Fast Stain
MATERIALS:
1. selected Mycobacterium species (Mycobacterium smegmatis)
2. non-acid fast organism (Staphylococcus epidermidis)
3. Ziehl-Neelsen carbolfuchsin
4. acid-alcohol
5. methylene blue
6. steaming apparatus
7. inoculating loop
8. scissor
PROCEDURE: Work in groups of 2 (two).
1. Set up the steaming apparatus; make sure the water is boiling slowly.
Microbiology Lab Manual,
2. Place a heat fixed bacterial smear of Mycobacterium smegmatis and Staphylococcus
epidermidis as a mixture on the steaming apparatus.
3. Cut bibulous paper to fit over the smear and place it on the slide.
4. Add carbolfuchsin (primary stain) to the paper on the smear.
5. Steam the slide for 5 minutes on the wire gauze of the steaming apparatus. Add dye
as needed to keep the paper wet during the steaming.
6. Wash the slide with water.
7. Add acid-alcohol decolorizer in drop wise fashion until the red dye stops running off
(or add acid-alcohol to the slide for 15 seconds).
8. Wash the slide with water.
9. Blot dry slide with bibulous paper.
10. Add methylene blue (counterstain) for 1 min.
11. Wash the slide with water.
12. Blot the slide dry with bibulous paper.
13. Observe the slide with 100X oil immersion.
Make Drawing
Questions
1. Name the genus which can be stained by acid-fast stain.
6. Which long chain fatty acid is present in the cell wall of genus Mycobacterium?
MOTILITY
OBJECTIVES:
Bacteria are complex organisms with a variety of structures. Flagella are used by organisms for
locomotion. Motility and the arrangement of flagella around the cell are important taxonomic
characteristics that are useful in characterizing bacteria. Flagella are long and thin that they are
hard to see.
Flagella Stain
This is the direct method which shows the presence of flagella. Flagella stain builds up dye
around the flagella until it is large enough in diameter to be seen with a microscope.
A-Monotrichous B-Lophotrichous
C-Amphitrichous D-Peritrichous
MATERIALS:
Motility
1. two organisms based upon availability any organism can be used Proteus vulgaris, E.
coli, Staphylococcus epidermidis, Streptococcus pyogenes
Non-motile Motile
Questions
1. Name the structure used for motility in both eukaryotes and prokaryotes.
OBJECTIVES:
The negative and capsule staining procedure is frequently used to examine capsule of bacteria.
Negative stain gets its name for two reasons. First, the background is stained instead of the
organism so the field of view looks like a photographic negative with light objects on a dark
background. Second, the stain used is an acidic dye with a negative charge that is repelled by the
negative cytoplasm of the bacterial cell.
Three stains are used for negative stain are Nigrosin, Congo red and India ink. Nigrosin and
Congo red are two acidic dyes. India ink is a neutral dye composed of particles too large to enter
a cell and does not have any charge on it. The ink fills in the background around the organism.
The background is dark while the cell is light. This reaction is not based on electrical charge,
just on particle size.
The capsule is a protective coating of the bacterial cell. It helps bacteria stick together and resist
phagocytosis. In capsule stain negative and simple stain procedures are combined to observe the
capsules. Typically an acidic stain such as Congo red or nigrosin, which stains the background,
and a basic stain which stains the bacteria are used in combination. The background will be dark
because of the negative stain and the organism will take basic stain. The capsule remains clear
since neither the acidic or basic stain can bind to capsule and appears as a white halo between the
bacteria and colored background.
The preparations for the negative stain and the capsule stains are very fragile. Negative staining
requires the use of either acidic dye or India ink. Bacteria are NOT heat fixed in this staining
technique as capsule is destroyed heat. Extra care must be taken to handle slides so that the
bacteria and stain do not wash off. Also, the bacteria are still alive so they can be infectious.
Diseases caused by encapsulated bacteria are S. pneumoniae causes pneumonia, otitis media and
meningitis, E. coli causes UTI, travelers diarrhea, meningitis and gastroenteritis, K. pneumoniae
Questions
1. When might one choose to perform the negative stain?
5. Name the bacteria and diseases caused by the bacteria which possess capsule.
OBJECTIVE
1. Label tryptic soy agar plates with station number and also label pretest on the 1st plate and
4. Gently press the diaphragm to the surface of the tryptic soy agar marked as pre-test.
6. Use diaphragm of the stethoscope to listen for breath (heart) sounds on your partner.
7. Gently press the diaphragm to the surface of the tryptic soy agar plate marked as post-test.
9. Incubate the agar plates for 24-48 hours at 370 C and read the results.
RESULT:
Questions:
1. What is a fomite?
3. Are you sterilizing the stethoscope diaphragm when you clean it with an alcohol wipe?
OBJECTIVES:
1. Discuss different biochemical processes occurring in bacteria and how these processes can
be used to identify bacteria.
2. Discuss the purpose and interpretation of each biochemical test.
3. Discuss substrate, enzyme and end products in each biochemical test.
5. Discuss the importance of positive and negative test results.
The staining procedures that were done in the previous labs allowed us to see some general
characteristics of bacteria. The size, shape and arrangement gave some hints about the type of
bacteria with which we were working. The Gram stain was the first step in classification.
However, it is impossible to identify a suspected pathogen without knowing the biochemical
characteristics of the bacterium.
The biochemical characteristics of a bacterium reflect the metabolic reactions occurring within
the cell. Each type of bacterium has its own set of biochemical reactions that are typical for that
organism. Therefore, organisms can be differentiated by biochemical reactions and classified
down to genus and species. There are many substrates that are utilized releasing products that
can be analyzed. We will be inoculating a variety of media with organisms to look at some of
the biochemical reactions for common organisms. The substrates, enzymes and products are to
be studied.
CARBOHYDRATE FERMENTATION
MATERIALS:
1. selected bacterial species: E. coli, S. epidermidis, B. subtilis, P. vulgaris
2. fermentation test tubes: lactose, sucrose and dextrose - 1 (one) each per person (12
tubes total for a group of 4)
DIGESTION OF STARCH
Starch is a complex polysaccharide composed of thousands of glucose molecules in straight and
branched chains. Some bacteria make the enzyme amylase which breaks down the
polysaccharide into smaller carbohydrates. The medium used to test for amylase is nutrient agar
that contains starch. Iodine is used to determine whether the organism can digest the starch in
the medium.
amylase
Starch Glucose subunits
Glucose subunits + iodine = clear zone around growth
MATERIALS:
1. selected bacterial species: E. coli, S. epidermidis, B. subtilis, P. vulgaris
2. starch agar plates - 1 plate per person
3. iodine solution
CATALASE TEST
MATERIALS:
1. selected bacterial species: E. coli, S. epidermidis, B. subtilis, P. vulgaris
2. 3% hydrogen peroxide solution
3. glass slides
Some bacteria produce the enzyme DNase which breaks long chains of DNA down into
nucleotides. The production of DNase is an identifying characteristic for certain bacteria. The
medium used to test for DNase contains DNA and methyl green. The long chains of DNA bind
the dye. If DNase is produced by an organism and the DNA is broken down, the DNA no longer
binds the dye. In the area of the agar where the DNase has broken down into nucleotides the
agar turns clear. A clear area around the bacterial growth indicates production of DNase. The
color change from green to clear is due to a pH change which occurs when the DNA is broken
down to nucleotides.
DNase
DNA Nucleotide subunit
MATERIALS:
1. selected bacterial species - E. coli, S. epidermidis, B. subtilis, P. vulgaris
2. DNase test agar - 1 per person
Indole production
The amino acid tryptophan is present in SIM agar as a source of pyruvic acid for organisms. If
tryptophan. Some bacteria produce the enzyme tryptophanase which breaks down the amino
acid tryptophan into indole, pyruvic acid and ammonia. Indole is a product with a putrid smell
that gives fecal material part of its characteristic odor. The pyruvic acid can be utilized in the
Krebs cycle to produce energy for the cell. The ammonia is given off. Indole can be visualized
by the addition of Kovac's reagent (this contains para-amino benzaldehyde which reacts with
indole). If indole is present, a red ring appears at the top of the tube. If no indole is produced,
the reagent sits at the top of the tube and remains an amber color (sometimes the reagent turns
green).
tryptophanase
Tryptophan indole + pyruvic acid + NH3
Indole + Kovacs reagent = Red ring on the surface
Motility
SIM agar may also be used to detect motile organisms. This is a semisolid agar that encourages
the movement of motile organisms away from the line of inoculation. Growth of non-motile
organisms is confined to the line of inoculation.
MATERIALS:
1. selected bacterial species - E. coli, S. epidermidis, B. subtilis, P. vulgaris
2. tubes of SIM medium - 1 per person
3. inoculating needle
UREASE TEST
Urea is an end product of protein metabolism in many organisms. The enzyme urease produced
by some bacteria digests urea into ammonia and carbon dioxide. If an organism is grown in
medium containing urea and if the organism makes urease, the urea is broken down as described.
The ammonia produced increases the pH of the medium, which also contains the pH indicator
phenol red. The pH indicator phenol red is affected by the increase in pH. The medium turns a
bright magenta (deep pink) when ammonia is released from urea.
Urease
Urea + H2 O NH3 + CO2 + H2 O
MATERIALS:
1. selected bacterial species - E. coli, S. epidermidis, B. subtilis, P. vulgaris
2. urea slants - 1 per person
Complete the following table by using A for acid, G for gas, a + for positive and a
- for negative results.
D S L
Proteus
Bacillus
S. epidermidis
E. coli
Serratia
Questions
1. List reasons for studying biochemical characteristics of bacteria.
3. What is the name of small test tube in carbohydrate fermentation test tube?
5. Name the enzyme which breaks down the polysaccharide into smaller carbohydrates?
6. Name the enzyme which breaks down hydrogen peroxide into water and oxygen?
11. What is the end product which causes blackening of the SIM agar?
13. Name the enzyme produced by the bacteria which hydrolyzed urea?
14. What is the name of pH indicator in urea agar and what is its function?
Since the natural histories of parasitic diseases differ in some important respects from those of
bacterial diseases, they merit a separate session to give you introductory laboratory experience
with parasites, the diseases they cause, and techniques used to diagnose them.
The distinguishing features of parasitic life are the close contact of the parasite with the host in
or on which it lives and its dependency on the host for life itself. This special association has led
to the evolution of three types of adaptations not found in the free-living relatives of the
parasites: loss of competency, special structures, and ecological ingenuity.
Parasites have become so dependent on their hosts for food and habitat that they now experience
a loss of competency to live independently. They usually require a specific host, and many have
lost their sensory and digestive functions; these are no longer important for their survival.
On the other hand, they have developed special structures and functions not possessed by their
free-living relatives that promote survival within the host. One example is special organs of
attachment hooklets and suckers. Parasites also have a tremendously increased reproductive
capacity, which compensates for the uncertainty in finding a new host. Tapeworms, for example,
have fantastically high rates of egg production, reaching up to 100,000 per day.
These three strategies promote survival and expansion of the species by providing greater
opportunities for finding and infecting new hosts, which is a continual problem for parasites.
Successful interruption of these cycles to prevent their completion is an important feature of
public health measures used to control diseases caused by parasites.
This exercise is designed to give you some practical experience with representative protozoan
and helminthic parasites and with clinical methods used in their diagnosis and control. Your
study will consist of these procedures:
1. As an introduction, you will have an opportunity to observe the movements and structure
of some living nonparasitic protozoans and worms often found in pond water.
The following classification of parasites will serve as a guide to the examples you will be
studying in this exercise. It is not a complete listing.
Protozoa
Protozoa, a subkingdom of the kingdom Protista, are unicellular eukaryotic organisms. They
usually reproduce by cell division and are classified mainly according to their means of
locomotion. Only one phyla, the Suctoria, which is closely related to the Ciliophora, does not
contain animal pathogens. The remaining is classified as follows:
Sarcodina
Members of this subphylum move and feed slowly by forming cytoplasmic projections known as
pseudopodia (false feet). They also form both trophozoites (vegetative form) and cysts
(resistant, resting cells). Parasitic members include the amoeba Entamoeba histolytica, which
causes amoebic dysentery. It ingests red blood cells and forms a four-nucleate cyst. It is also
found in animals. Other amoeba species found in humans, such as Entamoeba gingivalis, are
relatively harmless commensals.
Ciliophora
Members of this phylum have many short, hairlike cilia on their body surface that beat
rhythmically by bending to one side. Ciliophora is typified be genera Paramecium. Another
member, Balantidium coli, is a common parasite in swine. It possesses both cyst and trophozoite
form and can infect humans, causing serous results.
Mastigophora
These protozoans propel themselves with one or more long, whip-like flagella. Some have more
than one nucleus, and they usually produce cysts. Different species cause infection in the
intestine, vagina, blood, and tissues. Giardia lamblia causes a mild to severe diarrheal infection.
Trichomonas vaginalis is found in the urogenital region, where it causes a mild vaginitis in
women. Trypanosoma gambiense infects the blood via tsetse fly bites, causing trypanosomiasis,
or African sleeping sickness, in cattle and humans. Cattle and other ungulates serve as a
reservoir for this organism.
Sporozoa
Sporozoa are obligate, non-motile parasites with alternating stages: the sexual reproductive stage
is passed in the definitive insect host, and the asexual phase is passed in the intermediate human
Helminths (Worms)
Phylum Nematoda
Members of Nematoda (roundworms) occupy an important ecological niche since they are
present in large numbers in very diverse environments, including soil, fresh water, and seawater.
In contrast to the Platyhelminthes, these round, unsegmented worms are coelomate (have a body
cavity), and have a complete digestive tract and separate sexes. This phylum contains many
agents of animal, plant, and human parasitic diseases. Most require only one host and can pass
part of their life cycle as free-living larvae in the soil. Trichinella spiralis requires alternate
vertebrate hosts. Humans become infected when they ingest inadequately cooked meat, such as
pork or bear containing the larval forms in the muscles. Ascaris lumbricoides is probably the
most common worldwide of all the human helminths. Enterobius vermicularis causes pinworm,
a very common condition in children in the United States. Efforts to eradicate it have not been
very successful since pinworm causes little discomfort.
Definitions
Acoelomate. Without a true body cavity. Typical of members of the Phylum Platyhelminthes
(flatworms).
Cercaria. The last miracidium stage in which the larvae possess a tail.
Coelomate. With a true body cavity. Typical of members of the Phylum Nematoda
(roundworms).
Commensal. A relationship between two organisms in which one partner benefits from the
association and the other is unaffected.
Definitive host. The host in which the sexual reproduction of a parasite takes place.
Intermediate host. The host that is normally used by a parasite in the course of its life cycle and
in which it multiplies asexually but not sexually.
Merozoites. Schizont nuclei that become surrounded by cytoplasm and bud off as daughter cells
or merozoites.
Miracidium. A free-swimming ciliate larva that seeks out and penetrates a suitable intermediate
snail host, in which it develops into a sporocyst.
Proglottid. Any of the segments of a tapeworm formed in the neck region by a process of
strobilation (transverse fission).
Pseudopodia. Extensions of cytoplasm that aid in engulfing particles and functioning in motility
of amoeboid cells.
Schizont. A stage in the life cycle of Sporozoa in which the nucleus undergoes repeated nuclear
division without corresponding cell divisions.
Scolex. The head of a tapeworm, which is used for attaching to the hosts intestinal wall.
Sporocyst. A stage in the life cycle of certain protozoa in which two or more of the parasites are
enclosed within a common wall.
The malaria parasite life cycle involves two hosts. During a blood meal, a malaria-
infected female Anopheles mosquito inoculates sporozoites into the human host .
Sporozoites infect liver cells and mature into schizonts , which rupture and release
merozoites . (Of note, in P. vivax and P. ovale a dormant stage [hypnozoites] can
persist in the liver and cause relapses by invading the bloodstream weeks, or even years
later.) After this initial replication in the liver (exo-erythrocytic schizogony ), the
parasites undergo asexual multiplication in the erythrocytes (erythrocytic schizogony
). Merozoites infect red blood cells . The ring stage trophozoites mature into schizonts,
which rupture releasing merozoites . Some parasites differentiate into sexual
erythrocytic stages (gametocytes) . Blood stage parasites are responsible for the
clinical manifestations of the disease.
The gametocytes, male (microgametocytes) and female (macrogametocytes), are
ingested by an Anopheles mosquito during a blood meal . The parasites multiplication
in the mosquito is known as the sporogonic cycle . While in the mosquito's stomach,
the microgametes penetrate the macrogametes generating zygotes . The zygotes in
turn become motile and elongated (ookinetes) which invade the midgut wall of the
mosquito where they develop into oocysts . The oocysts grow, rupture, and release
sporozoites , which make their way to the mosquito's salivary glands. Inoculation of
the sporozoites into a new human host perpetuates the malaria life cycle.
The only known definitive hosts for Toxoplasma gondii are members of family Felidae (domestic cats
and their relatives). Unsporulated oocysts are shed in the cats feces . Although oocysts are usually
only shed for 1-2 weeks, large numbers may be shed. Oocysts take 1-5 days to sporulate in the
environment and become infective. Intermediate hosts in nature (including birds and rodents) become
infected after ingesting soil, water or plant material contaminated with oocysts . Oocysts transform
into tachyzoites shortly after ingestion. These tachyzoites localize in neural and muscle tissue and
develop into tissue cyst bradyzoites . Cats become infected after consuming intermediate hosts
harboring tissue cysts . Cats may also become infected directly by ingestion of sporulated oocysts.
Animals bred for human consumption and wild game may also become infected with tissue cysts after
ingestion of sporulated oocysts in the environment . Humans can become infected by any of several
routes:
In the human host, the parasites form tissue cysts, most commonly in skeletal muscle, myocardium,
brain, and eyes; these cysts may remain throughout the life of the host. Diagnosis is usually achieved by
serology, although tissue cysts may be observed in stained biopsy specimens . Diagnosis of congenital
infections can be achieved by detecting T. gondii DNA in amniotic fluid using molecular methods such as
PCR .
Microorganisms require certain basic nutrients and physical factors for the sustenance of life.
However, their specific requirements vary greatly. Understanding these needs is essential for the
successful growth of microorganisms in the laboratory.
Special purpose media are available for the functions such as:
SELECTIVE MEDIA
A selective medium is used to select specific group of bacteria. A selective medium contains
chemical substances that inhibit the growth of one type of bacteria while allowing the growth of
another type of bacteria, thus facilitating bacterial isolation.
DEFFERENTIAL MEDIA
A differential medium can distinguish between morphologically and biochemically related
groups of bacteria. A differential medium contains chemical compounds that following
inoculation produces a characteristic change in the appearance of bacterial growth and /or the
medium surrounding the colonies, which permits differentiation.
ENRICHED MEDIA
An enriched medium has been supplemented with highly nutritious material, such as blood,
serum or yeast extract, for the purpose of growing fastidious bacteria.
OBJECTIVES:
1. Differentiate between Staphylococcus aureus and Staphylococcus epidermidis colony
characteristics
2. Differentiate pathogenic Staphylococci from non-pathogenic Staphylococci by
biochemical tests.
3. Differentiate Staphylococci from Streptococci.
4. Discuss different tests performed to differentiate between Staphylococcus aureus and
Staphylococcus epidermidis.
5. Interpret data from the biochemical tests needed for diagnosis of Staphylococci.
5. Learn the name of the diseases caused by Staphylococcus aureus and Staphylococcus
epidermidis.
The Staphylococci are Gram (+) cocci occur in clusters, inhabits human skin and nose. Two
representative organisms from this group are S. aureus and S. epidermidis. The colonies of S.
aureus are round and golden yellow in color. The colonies of S. epidermidis are white color
small pinpoint colonies. They are salt tolerant (NaCl to 10%).
S. aureus causes food poisoning, acute bacterial endocarditis, toxic shock syndrome, boils,
wound infections, osteomyelitis and meningitis. S. epidermidis is a normal flora of the skin. It is
not usually involved in disease under normal conditions. Under certain conditions, S.
epidermidis can be involved in sub-acute endocarditis, catheter infections and prosthetic joint
infections.
Coagulase Test
The pathogenic Staphylococci produce coagulase. S. aureus makes coagulase and is a pathogen.
S. epidermidis does not produce coagulase and is not considered a pathogen. Plasma clots in the
presence of coagulase. If plasma is inoculated with an organism and a clot forms, then the
organism is producing coagulase and, it is a pathogen.
The two tests for pathogenic Staphylococci are the fermentation of mannitol and the production
of coagulase. Both S. epidermidis and S. aureus are catalase (+)
MATERIALS:
1. cotton swabs
2. sterile saline
3. Mannitol-salt agar plates (2 per student)
4. cultures of S. aureus and S. epidermidis
MATERIALS:
1. tubes containing 0.5ml coagulase plasma (one per group of two students).
2. cultures of S. aureus and S. epidermidis
3. Pasteur pipettes and bulbs
MATERIALS:
1. Culture plates
2. 3% hydrogen peroxide
PROCEDURE:
1. Add two to three drops of hydrogen peroxide in each plate
Staphylococcus aureus
Staphylococcus
epidermidis
Questions
OBJECTIVES:
1. Isolate Streptococci from various parts of the upper respiratory tract.
2. Recognize and interpret the different types of hemolysis (alpha, beta, and gamma).
3. Discuss diseases caused by genus Streptococci.
4. Discuss the Streptococcus viridans and their relation to disease of the oral cavity.
5. Use the biochemical tests necessary to select and identify Streptococci.
6. Discuss and use appropriate Universal Precautions for Blood Borne Pathogens.
The Streptococci are a group of Gram (+) many are facultative anaerobic cocci that form chains.
The organisms grow best on enriched media (e.g. blood agar) in an atmosphere of 5-10% CO2.
Many of the streptococci are normal flora of the skin, upper respiratory tract, mouth and the
intestines. The pathogen in this S. pyogenes causes strep throat, necrotizing fasciitis, scarlet
fever, rheumatic fever, impetigo, erysipelas, toxic shock syndrome and glomerulonephritis. The
streptococci are classified by the hemolytic reaction on blood agar plates. The organisms can be
partially classified on the basis of hemolysis.
() Alpha-hemolysis
Organisms are alpha-hemolytic if the red blood cells are incompletely destroyed and produce
green zone around the colony. S. mitis and S. pneumoniae are alpha-hemolytic.
() Beta- hemolysis
Organisms are beta-hemolytic if the blood cells in the agar are completely destroyed by the
hemolysin produced by the bacteria. A clearing is evident around the colonies of beta-hemolytic
organisms. S. pyogenes and S. agalactiae is beta-hemolytic.
() Gamma- hemolysis
The organisms that do not hemolyze red blood cells at all are gamma-hemolytic. S. lactis and S.
faecalis are gamma-hemolytic.
In this exercise, organisms from the upper respiratory tract will be examined. Normal flora of the
upper respiratory tract include - hemolytic Streptococci, Neisseria speciesStaphylococcus
aureus and other Staphylococcus species, - hemolytic Streptococci, and - hemolytic
Streptococci (in a few people). A large number of -hemolytic Streptococci indicate infection or
carrier state.
Some of the streptococci found in the oral cavity contribute to tooth decay. The viridans group
of streptococci such as S. mutans, S. mitis and S. salivarius produce acid which attacks tooth
enamel. Mitis-salivarius medium can be used to grow these organisms. The medium contains
tellurite which selects for the viridans group of streptococci. The colonies will be blue. This
medium is not differential. S. mitis produces pinpoint colonies while S. salivarius produces
gumdrop shaped colonies.
MATERIALS:
1. tongue depressors
2. sterile swabs
3. blood agar plates - one (1) per person.
4. protective gown, goggles, gloves, and mask obtained from an instructor.
MATERIALS:
1. cotton swabs
2. Mitis-Salivarius medium (one plate per person)
Blood Agar
Streptococcus pneumoniae
Streptococcus pyogenes
Streptococcus mitis
Streptococcus salivarius
Questions
1. Describe genus Streptococcus.
3. Describe different types of hemolysis and name the species which cause different types of hemolysis.
4. According to OSHA guideline what should be used while working with body fluids?
Bloodborne Pathogens means pathogenic organisms that are present in human blood and can cause disease in
humans. These pathogens include but are not limited to hepatitis B virus (HBV) and human immunodeficiency
virus (HIV).
Other Potentially Infectious Materials means the following body fluids: semen, vaginal secretions, cerebrospinal
fluid, synovial fluid, pericardial fluid, peritoneal fluid, amniotic fluid, saliva, any body fluid visibly
contaminated with blood and ALL body fluids in situations where it is difficult to differentiate between body
fluids.
Universal Precautions: According to the concept of Universal Precautions, all human blood and certain human
body fluids are treated as if known to be infectious for HIV, HBV, and other bloodborne pathogens.
WORKING IN GROUPS-
a. All members of the group must wear lab coats and gloves. Goggles and masks are available.
b. Obtain and test a urine sample according to the procedure in the lab manual.
c. Wipe up any spills immediately and place the contaminated toweling in the biohazard bag.
Disinfect the area and materials with bleach. Dispose soiled gloves into the biohazard bag.
Wash hands and re-glove with sterile gloves.
WORKING IN GROUPS-
a. All members of the group, not giving blood, will wear lab coats, gloves, masks and goggles.
b. Students will work ONLY with their own blood.
_______________________________
Print Name:
_______________________________ ___________________
Signature Date
OBJECTIVES:
Some of the Neisseria such as N. sicca, N. mucosa and N. subflava are normal flora of the upper respiratory
tract. Therefore, Gram (-) diplococci in the oropharynx can be normal. The two pathogens in this group are N.
meningitidis and N. gonorrhoeae which cause meningitis and gonorrhea respectively. Ten to thirty percent of
healthy asymptomatic individuals are carriers of N. meningitidis. These organisms require enriched media to
grow and prefer a 5-10% CO2 atmosphere. The Neisseria produce oxidase. The production of oxidase is
observed when tetramethylparaphenylene-diamine hydrochloride (oxidase reagent) is added directly to colonies
on a chocolate agar plate. This dye is an artificial electron acceptor instead of oxygen. The reagent is colorless
when reduced and when oxidized it turns purple and eventually black.
A non-selective, enriched growth medium used to grow genus Neisseria is called chocolate agar. This medium
contains hemolyzed sheep red blood cells and agar. Hemolyzed RBC's leak out extra nutrients. Because this
medium is so enriched almost any organism will grow on it, and this medium is not considered selective. There
is no characteristic of the medium to make it differential either.
Thayer-Martin Selective Agar is a selective medium for culturing and primarily isolating pathogenic Neisseria
gonorrhoeae and Neisseria meningitidis from specimens. It contains Chocolate II Agar with vancomycin,
colistin and nystatin, it is formulated to minimize the overgrowth of gonococci and meningococci by
contaminants, to suppress the growth of saprophytic Neisseria species and to enhance the growth of pathogenic
Neisseria.
Typical colonial morphology on Thayer-Martin Selective Agar is as follows:
Neisseria gonorrhoeae ..................Small, grayish-white to colorless, mucoid
Neisseria meningitidis ..................Medium to large, blue-gray, mucoid
MATERIALS:
1. cotton swab
2. chocolate agar plate - neither selective or differential - (one per person)
3. tongue depressor
Neisseria
Questions
1. Describe genus Neisseria
3. Name the species of genus Neisseria present in upper respiratory tract as normal flora.
OBJECTIVES:
The Gram (-) non-sporeforming rods are called the enteric bacteria. The majority of them are normal flora of
the gastrointestinal tract. They belong to the family Enterobacteriaceae. The major pathogens are Salmonella
typhi causes typhoid fever, Shigella causes shigellosis or bacillary dysentery, Escherichia coli cause travelers
diarrhea, UTI, diarrhea in infants and Proteus causes UTI.
Isolation of organisms from the GI tract can be difficult because the gut has a wide variety of organisms. The
enteric bacteria can be isolated on media that are both selective and differential.
MacConkey Agar
It is a selective medium, selective agent is bile salt and crystal violet inhibits Gram-positive cocci and allow
Gram-negative organism to grow. It is also a differential medium, differential agent is lactose. Lactose
fermentation causes drop in pH around the colony and color change in the pH indicator neutral red. Lactose
fermenting organisms take up the dyes and form pigmented (pink to brick red) colonies. The colonies of the
non-fermenters are colorless.
IMViC Test
A useful series of four tests called the IMViC Test is used to further identify the enterics. The "I" stands for
indole. The "M" stands for methyl red. The "V" stands for Vogues-Proskauer. The "i" aids in pronunciation.
The "C" stands for citrate. The indole test is done as previously described in the biochemistry experiment.
Indole production
In indole broth amino acid tryptophan is present. Some bacteria produce the enzyme tryptophanase which
breaks down the amino acid tryptophan into indole, pyruvic acid and ammonia. Indole is a product with a
putrid smell that gives fecal material part of its characteristic odor. The pyruvic acid can be utilized in the
Krebs cycle to produce energy for the cell. The ammonia is given off. Indole can be visualized by the addition
of Kovac's reagent (this contains para-amino benzaldehyde which reacts with indole). If indole is present, a red
ring appears at the top of the tube. If no indole is produced, the reagent sits at the top of the tube and remains
an amber color (sometimes the reagent turns green).
MR test
Glucose pyruvic acid
Pyruvic acid lactic acid, acetic acid and formic acid (mixed acids)
VP test
Glucose pyruvic acid
ISOLATION OF ENTERICS
MATERIALS:
1. MacConkey agar three plates
2. EMB agar three plates
3. Pure culture of E. coli, Proteus and Enterobacter aerogenes.
MATERIALS:
1. Pure cultures of E. coli, Proteus and Enterobacter aerogenes.
2. TSIA (one tube per person)
E. coli
Proteus
Enterobacter aerogenes
Microorganism Slant color Butt color Sugar Gas production H2S production
fermentation
E. coli
Proteus
Enterobacter
aerogenes
Sugar fermentation: red/yellow = Glucose, yellow/yellow = Lactose and or Sucrose and red/red = no
reaction
IMViC Reactions
Microorganism I MR VP C
E. coli
Proteus
Enterobacter
aerogenes
2. Describe:
MacConkey agar
6. In IMViC series
a. I stands for
b. M stands for
c. Vi stands for
d. C stands for
7. Why is it unlikely that an organism will be positive for both the methyl red and Voges-Proskauer tests?
1. Your unknown is worth 30 points when completed in full. If you do not complete all of your
unknown work, or if you are incorrect on some of it, you may receive partial points, as described
below.
4. Your instructor must observe your gram stain to receive credit. If you are correct in form,
morphology and arrangement, you may continue to the next step. If you are not, you will be given a
second chance (2 points will be deducted). You may need to make another gram stain, or just
reevaluate what you see. Dont just guess!
5. When you have determined morphology, arrangement and gram reaction correctly, you will choose
appropriate biochemical tests.
Choose appropriate biochemical media relevant to solve your unknown organism.
Label all media with your station number and your known number.
Inoculate biochemical tests and incubate them.
You may use your lab book and textbook as references. Do not copy material from lab notes word
for word.
Read biochemical reactions and record results.
Using the biochemical evidence and the information determined from observing the growth,
morphology, and gram reaction determine the genus and species of your unknown.
6. Show your instructor your results. If you are correct, your paper will be signed off. If you are
incorrect, you will be given a second chance. Think, do not guess.
I. Introduction
This should include the identified genus and species. Describe its habitat, mode of transmission, growth
requirements and name of diseases found in humans. The report should include if appropriate issues
the organism can cause in the environment that can also impact human health.
Methods: Without copying from on-line lab manual. List all tests that were performed and the relevance
of each test in determining the name of the unknown specimen. Do Not list step by step instructions on
how to perform the tests (i.e. Gram stain was performed to determine .; Do not write out how to
perform each section of a gram stain).
III. Results
Present your data in table format listing the tests performed, your observations and the results. For example:
Gram Stain Pink rods Gram (-) bacillus
Also create is a dichotomous key (division into two parts) formatting the correct order of tests and the
identification of the organism.
Your conclusion should include application of the techniques used to real world situations that relate to your
identified organism. Your discussion and conclusion should be reported in a concise manner, and there must
be correlation to what is presented in the introduction.
V. Format
You report must be typed and printed with a cover sheet including the name of the organism identified and all
of the names of those in your lab group. The worksheet must be attached to the end of the report.
The report itself should be two to four pages double spaced, 12 size font, spellings and grammar counts. In
your report include all headings. If you include pictures you have taken of slides in the report it references
the slide for the color then the report must be submitted in color. If you will not follow the format points
will be deducted.
VI. References
Your report needs to include two references in addition to the textbook and/or lab manual. Wikipedia is Not
an acceptable reference. If you use Wikipedia must go to read and then use the correct cited reference. You
cannot just list Wikipedia.
Unknown Number
Biochemical test
Food Microbiology
OBJECTIVE:
1. To make yogurt.
2. To understand the effects of fermentation on food.
Microbes are important in food production including foods such as beer, wine, cheese, yogurt,
sour cream, buttermilk. Yogurt is produced by the fermentation of warm milk by Lactobacillus
bulgaricus or Lactobacillus acidophilus and Streptococcus thermophilus. These bacteria grow
at 40-45oC. The milk is modified as a result of anaerobic fermentation which produces a
mixture of organic wastes including lactic acid and various other byproducts. These curdle the
milk and solidify the proteins as well as giving yogurt its unique flavor. Food fermentation
involves the use of starter cultures that contain known organisms to carry out specific and
reproducible fermentation reactions.
Specific starter bacteria are added to the milk. Bacteria ferment milk sugar lactose into lactic
acid. The presence of the acid lowers the pH of the milk, making it acidic. As the pH is lowered,
the proteins casein in the milk begins to coagulate, giving firm texture to yogurt.
Yogurt is the best known food to contain healthy bacteria. Commercial yogurts are now being
enhanced with probiotics specifically to aid intestinal function to produce healthy digestion.
Yogurt is a concentrated protein product that has been enriched by additional enzymes and
vitamins produced by the microbes.
MATERIALS:
1. 1% milk 100 ml
2. One teaspoon of yogurt to use as starter culture
3. Steaming apparatus (Bunsen burner, ring stand, beaker and wire gauze)
4. Plastic container with lid
5. Spoon
6. Thermometer
PROCEDURE: Work in group
1. Label plastic container with your station number.
2. Put milk in the beaker and heat it up to 700 C to kill any bacteria present.
3. Add a teaspoon of starter culture in the plastic container.
4. Cool milk to 40- 500 C.
5. Add milk to plastic container and stir it.
6. Close plastic container with lid and incubate it at 400 C for four to six hours.
1. Define probiotics
There are a variety of physical methods for control of microorganisms. Direct heat is used when
wire loops are flamed. Pressurized steam is used in autoclaves to sterilize a wide variety of
items, from microbiological media to linens. Ultra-violet light is used to sterilize air and
surfaces. Filters are used to sterilize liquids like antibiotics and vaccines which cannot be
heated. In this lab we will look at the effects of heat and ultra-violet light on different organisms.
HEAT:
Boiling water is effective in destroying organisms. This moist heat method of sterilization
causes conformational changes in proteins. Once enzymes or structural proteins change shape
the function is lost, therefore, the organisms die. Some bacteria are more sensitive to heat than
others. In this experiment organisms will be exposed to heat for different amounts of time. The
survivors will eventually grow in the broth. We will look for survivors by examining broth
cultures for turbidity.
ULTRA-VIOLET LIGHT:
Ultra-violet light at 265 nanometers causes the production of thymine dimers in the DNA of the
microorganism. These dimers prevent the transcription of the DNA into a viable mRNA.
Therefore protein synthesis is also interrupted which leads to lethal mutation. Without protein
synthesis organisms die. Sometimes the mutation is not lethal in which bacteria replicate but
only one characteristic of the organism is altered. We will examine the effect of U.V. light on
Serratia marcescens.
ATCCTTAGTTACG
TAGGAATCAATGC
UV
ATCCTTAGTTACG
TAGGAATCAATGC
Questions
1. Name different physical method used to control growth of microorganisms.
OBJECTIVES:
Many chemical agents inhibit bacteria. Antiseptics are used on living tissue to kill
microorganisms. Disinfectants are used on inanimate objects to remove and inactivate
organisms. A wide variety of commercial products that will inhibit microorganisms are
available.
ANTIBIOTICS
Antibiotics are natural substances produced by one microorganism (primarily molds) that inhibit
another organism or group of organisms (primarily bacteria). Sulfonamides were first used in
1935 followed by penicillin in 1940. Since then many useful antibiotics have been discovered,
such as chloramphenicol, tetracycline, and streptomycin.
The effectiveness of antibiotics is determined by the Standardized Disk Susceptibility Test, also
known as the Kirby-Bauer Method of Antibiotic Sensitivity Testing. A special medium called
Mueller-Hinton agar is used for antibiotic sensitivity testing because it gives reproducible
results and does not inhibit Sulfonamides. The agar is inoculated with a lawn of bacteria. Paper
discs containing known amounts of antibiotics are placed on the plates. After incubation, the
plates are observed for zones of inhibition of growth of the organism. The size of the zone of
inhibition is measured and compared to a table of known sizes of zones. The sensitivity of the
bacterium to a particular antibiotic can be determined. This method is quantitative due to the
fact that a standardized medium is used and the paper discs contain a known concentration of
antibiotic.
I. GENERAL
A. Fungi are chemoheterotrophs that require organic compounds for energy and carbon. Their
cell wall is made of a polysaccharide chitin. They are absorptive heterotrophs that they secret
exoenzymes into the environment, and then absorb the digested nutrients.
Majority are saprophytes that decompose dead or decaying organic matter. Some are
parasites of humans, animals, and plants.
B. Includes Molds ( filamentous), Yeasts ( unicellular) and dimorphic (both mold and yeast)
Division into these categories is based on overall appearance and how they grow.
C. Nutritional Adaptations
These characteristics allow fungi to grow on unlikely substrates such as painted walls
and shoe leather.
1. Fungi usually grow better in an acidic pH (5.0) with high sugar concentrations.
2. Most Molds are aerobic, so they grow on surfaces rather than through a substrate. Yeasts
are facultative anaerobes.
3. Most fungi are more resistant to osmotic pressures than bacteria are; most fungi are
therefore able to grow in high sugar or salt concentrations.
4. Fungi are capable of growing on substances with very low moisture content, generally
too low to support the growth of bacteria.
6. Fungi are capable of using complex carbohydrates, such as wood, that most bacteria
cannot metabolize.
D. Fungi are found in almost any climate but prefer warm, moist environments.
1. They are non-filamentous unicellular fungi that are typically spherical or oval.
3. Reproduce by Budding parent cell forms a protuberance (bud) that elongates, the
parent cells nucleus divides and one nucleus migrates into the bud. Cell wall material
laid down between bud and parent cell, bud breaks off.
Molds (multicellular)-
1. The body (thallus) consists of long filaments of cells joined together called
hyphae (hypha).
2. Hyphae grow by elongating tips. Hyphae grow, intertwine and form a mass called
mycelium.
Dimorphic Fungi
2. Dimorphic fungi have both Mold and Yeast life cycle stages.
These are asexual spores formed within a sac (sporangium) at the end of aerial
hyphae called sporangiophores. A sporangium can contain hundreds of
sporangiospores.
B. Basidiomycota Club fungi- Common name is derived from the shape of the
basidium that bears the sexual basidiospores; includes mushrooms.
1. They are imperfect because they have not yet been shown to produce sexual spores.
(Asexual reproduction only)
Fungal diseases
Name Mode of transmission Disease
Blastomyces dermatitidis Inhalation of dust carrying Cutaneous blastomycosis; Pulmonary
fungal spores blastomycosis
Trichophyton spp. spores shed in the Dermatophytoses
Microsporum spp., environment by infected Tinea pedis "athletes foot"
Epidermophyton individuals Tinea cruris " jock- itch"
Tinea unguium
Tinea capitis
Tinea barbae
Malassezia furfur Normal microbiota on skin Tinea versicolor (Pityriasis versicolor);
dandruff; seborrheic dermatitis
Sporothrix schenckii Thorn pricks, wood splinters Sporotrichosis "Rose-Gardners disease"
Aspergillus spp. contamination from spores Aspergillosis
Carcinogenic mycotoxin causes
hepatocellular carcinoma
Cryptococcus neoformans Inhalation of spores from Cryptococcal meningitis in AIDS patients.
contaminated pigeon or
chicken droppings
Coccidioides immitis Inhalation of spores Coccidioidomycosis (Valley fever)
Histoplasma capsulatum Inhalation of spores near Histoplasmosis (Spelunker's disease)
bird droppings
Pneumocystis jirovecii Inhalation of spores Pneumocystis pneumonia in AIDS patients.
(carinii)
Candida albicans opportunistic infection, oral thrush, vaginal thrush, Candidal
sexually transmitted onychomycosis, Candidal paronychia, Diaper
candidiasis, Congenital cutaneous
candidiasis, Perianal candidiasis,
Piedra "Trichosporosis" Fungal growth on hair