Introduction

Microbial species in a common environment often compete with other for

resources by inhibiting each others growth; some do this by producing
complex chemicals called antibiotics.

Antibiotics are complex chemical substances capable of inhibiting the growth

of microorganisms (such as bacteria) and are produced naturally by other
microorganisms such as moulds, bacteria and viruses as well as being
produced by chemical synthesis.

Some antibiotics are used in food preservation and non-medically in animal

feeds. But more commonly in medicine, typically against infections or
diseases and are however chemically altered to increase their effectiveness.
Antibiotics can be described as broad spectrum: this is when the antibiotic
acts on a wide range of gram positive bacteria (see later notes) or narrow
spectrum where the antibiotic is more specific.

Antibiotics affect processes characteristic to prokaryotes. Their action may be

described as either bacteriostatic where they stop or slow reproduction or
bacteriocidal if they kill the microbe. A test to determine the effectiveness of
antibiotic therapy against micro-organisms that have been isolated and
identified can be performed1. This is known as antibiotic sensitivity test.
Antibiotic sensitivity tests are vital in determining the appropriate drug
therapy for infections2. By finding the antibiotic that works the fastest and
kills the largest number of bacterial colonies, the most effective treatment
can be established. Antibiotic sensitivity tests can be carried out in various
ways but perhaps the most common is the disk plate method. In this
procedure an agar plate of a suitable medium is heavily inoculated with the
infecting microorganism whose sensitivity is in question3, antibiotic discs of
various concentrations or of different chemical class are placed on the agar.
The plates are then incubated and the diameters of the inhibition zones are
measured after a specific time interval (please see diagram sheets 1 and
2 for the typical results of this test).

Antibiotics have varied modes of action and are usually categorized by their
chemical class. Beta-lactam is the chemical name for antibiotics that have a
chemical/lactam ring. This extensive family includes the penicillins4

The natural action of penicillin was first observed in 1928 by British

bacteriologist Sir Alexander Fleming when he noticed the bactericidal
effects of a secretion from Penicillim notatum on colonies of Staphylococcus
bacteria. However it wasn’t until 1941 that Ernst Chain, Sir Howard Florey
and other scientists purified the substance and established its effectiveness;
allowing it to be used successfully in the treatment of infection through
injections (as it is inactivated by digestive acids in the stomach5).
1) http://www.umm.edu/ency/article/003744.htm
2) Same as above
3) Pg. 233, Microbiology in Health and Disease, M.Frobisher & R. Fuerst, 13th
Edition
4) Pg. 114, Medicines a Comprehensive guide, 3rd Edition
5) Pg. 111, same as above.
1) http://www.umm.edu/ency/article/003744.htm
2) Same as above
3) Pg. 233, Microbiology in Health and Disease, M.Frobisher & R. Fuerst, 13th
Edition
4) Pg. 114, Medicines a Comprehensive guide, 3rd Edition
5) Pg. 111, same as above.
Penicillin is a narrow spectrum antibiotic and acts by inhibiting the cross-
linking in the peptidoglycan (the main component of the cell wall in
bacteria6). This instigates abnormal growth for the newly produced cells
which in turn renders them ineffective at maintaining their cell wall rigidity.
This consequently, may lead to osmotic lysis7, thus establishing penicillin’s
action as time dependent and bactericidal. Pathogens such as pneumocci,
streptococci, gonococci and meningococci are particularly susceptible to the
antibiotic. Although penicillin is extremely effective when it does work, many
organisms are resistant to it (due the narrowness of its antibacterial
spectrum). Microbes such as certain strains of e.coli can release an enzyme
called penicillinase, which inhibits the action of the antibiotic. Hence the
search for broader spectrum antibiotics began. This led to the discovery of
streptomycin.

Streptomycin is produced by a soil organism called Streptomyces griseus and

was originally isolated by Selman A. Waksman and Albert Schatz8 in 1947 in
the treatment of tuberculosis. This broad-spectrum antibiotic belongs to the
chemical class of amino glycosides-which as the name suggests are
composed of amino sugars linked by glycosidic bonds to various bases. These
are rapidly bactericidal and act on various bacterial functions but their
primary target is protein synthesis9. For example low levels of streptomycin
cause misreading of the mRNA, while high levels completely inhibit protein
synthesis10, therefore establishing streptomycin as a concentration
dependent inhibitor. Streptomycin is particularly important because it inhibits
many organisms resistant to the sulphonamides and penicillin11. However it’s
broad and perhaps overuse has also resulted in a wide spread resistance to
the antibiotic, hence it is used in combination with other drugs such as
ethambutol.

As mentioned earlier antibiotics only affect processes characteristic to

prokaryotes, their mode of action is defined by which classification of bacteria
(gram positive and/or gram negative) they are effective against.
Classification of the bacteria is dependent on their reaction the Gram staining
technique, where a fixed bacterial smear is subjected to a number of
solutions including: crystal violet, iodine solution, ethanol/alcohol & safranin.

Gram positive bacteria appear bluish purple under a light microscope as they
take up the crystal violet stain and remain unaffected by the rest of the
process12. Examples include staphylococcus and bacillus bacteria;
characteristics common to these include two basic layers, a plasma
membrane and a thick layer of peptidoglycan. Typically these bacteria are
particularly sensitive to antibiotics such as penicillin and sulphonamides.

6) http://helios.bto.ed.ac.uk/bto/microbes/penicill.html
7) Same as above
8) http://print.factmonster.com/ce6/sci/A0846951.html
9)http://www.bmb.leeds.ac.uk/mbiology/ugteach/dental/antibiotics/dantibiobact/p
rotag.html
10) Pg. 394, Bacteria in Biology, Biotechnology & Medicine, Paul Singleton
11) Pg. 476, Microbiology, 4th Edition, M.J.Pelczcar, R.D.Reid and E.C.S Chan
12)Pg. 592, Biology, 2nd Edition, Ann Fullick, Heinemann Educational Publishers
2000
Gram negative bacteria such as salmonella, haemophilus and escherichia
coli, appear to red under a light microscope, as any crystal violet which does
bind to the bacterial smear is readily decolorised by the alcohol and replaced
with red safranin dye. Gram negative bacteria have thinner walls containing
less peptidoglycan, and are usually sensitive to broad-spectrum antibiotics
such as streptomycin.

E.coli is the abbreviated name of bacterium in the family enterobacteriaceae

named escherichia (genus) coli (species) 13, which are facultatively anaerobic
gram negative rods. Non pathogenic E.coli lives symbiotically as part of the
normal microflora of the intestine in humans & other and other animals14 in
which it provides sources of vitamin K and B complex vitamins. However it
only makes up a very small proportion of the bacterial content found inside
the intestines. Physiologically E.coli is versatile and well adapted to its
characteristic habitats15 as it is able to grow in the presence or absence of
oxygen since it is able to utilize nitrates and nitrites16.
In part this acclimatizes E.coli to its intestinal (anaerobic) and its extra
intestinal (aerobic and anaerobic) habitats.

However, despite the beneficial nature of non-pathogenic E.coli, pathogenic

E.coli (which is genetically different) is responsible for many infections in
humans such as urinary tract infections (UTI), neonatal meningitis and
intestinal diseases (gastroenteritis). These three diseases depend on a
specific array of virulence determinants17. In other words they are caused by
various strains of pathogenic E.coli.

For this experiment a pure culture of non-pathogenic E.coli in nutrient broth

was isolated and plated by 2 different methods: Spread and Pour plating.

Spread plating (a quantitative plating method) is otherwise known as lawn

plating and is made by spreading a small volume of liquid inoculum over the
surface of a solid medium by means of a sterile L-shaped glass rod (a
“spreader”)18, upon incubation a “lawn” of confluent growth can be seen on
the surface of the agar. Thus it can be said that this technique is convenient
for cultures that may respire aerobically.

Pour plating is also a quantitative method of plating and is scientifically

known as “plating out”. This technique introduces the sample into a tube of
melted warm (45ºC) agar culture medium which is mixed thoroughly and is
then poured into a petri plate19. Alternatively a small volume of the liquid

13) http://people.ku.edu/~jbrown/ecoli.html
14) Pg. 448, Bacteria in Biology, Biotechnology & Medicine, 5th Edition, Paul
Singleton
15) http://www.bact.wise.edu/Bact330/lectureecoli
16) Same as above
17) http://www.bact.wise.edu/Bact330/lectureecoli
18) Pg. 369, Bacteria in Biology, Biotechnology & Medicine, 5th Edition, Paul
Singleton
19)Pg. 376, Microbes in Action, A Laboratory Manual of Microbiology, 4th Edition,
Seeley, Vandemark, Lee
20) Pg. 376, Bacteria in Biology, Biotechnology & Medicine, 5th Edition, Paul
Singleton
inoculum is pipetted into a sterile petri dish via aseptic techniques. The
melted warm agar is then poured into the plate and the dish is gently swirled,
three or four times in each of the three directions. On incubation colonies
develop within as well as on the medium20.

13) http://people.ku.edu/~jbrown/ecoli.html
14) Pg. 448, Bacteria in Biology, Biotechnology & Medicine, 5th Edition, Paul
Singleton
15) http://www.bact.wise.edu/Bact330/lectureecoli
16) Same as above
17) http://www.bact.wise.edu/Bact330/lectureecoli
18) Pg. 369, Bacteria in Biology, Biotechnology & Medicine, 5th Edition, Paul
Singleton
19)Pg. 376, Microbes in Action, A Laboratory Manual of Microbiology, 4th Edition,
Seeley, Vandemark, Lee
20) Pg. 376, Bacteria in Biology, Biotechnology & Medicine, 5th Edition, Paul
Singleton
Hypothesis

It has been noted that spread plating the bacteria will ensure an even
distribution of confluent growth across the plate. Hence the hypothesis to be
tested is that the spread plating technique will aid the antibiotic’s
effectiveness by exerting a noticeable effect on the diameter of the zone of
inhibition

Null Hypothesis (See Statistical test for relevance)

There will be no significant difference between the ability of the two

techniques to aid the antibiotic’s effectiveness. Hence no noticeable effect
will be exerted on diameter of the zone of inhibition.

Independent Variable

Culture technique used.

This will be either spread or pour plating, and an equal number of plates
will be created by each technique.

Dependent Variable

Diameter of the inhibition zone

This will be measured after incubation of 24 hours and should be fairly
standardized as the antibiotic and concentration used in each technique is
undeviating.

Controlled Variables

• Temperature of the incubator

It is fundamental that the temperature of the incubator is controlled as
this can effect the rate of bacterial growth. For any given bacterium there
are maximum and minimum temperatures beyond which growth will not
occur. Penicillin (one of the antibiotics to be used in the pilot study) is a
time dependent bacteriocidal antibiotic that interferes with bacterial
growth-hence the rate of bacterial growth is influential to the final result.
Therefore to ensure that the rate of growth is uniform in all samples; the
agar plates will be incubated in an oven at a constant temperature of
35ºC.

• Nutrient content of agar

A solid medium (enriched with the appropriate nutrients that the bacteria
will require to grow on) will be used in this experiment. This will be made
from nutrient agar of the same concentration of dried tablets in an equal
volume of water.
 • Temperature of the water bath
An electric water bath will be used to prevent the nutrient agar from
solidifying before it is plated. This will be kept at a constant temperature
of 45ºC.

• Volume of Bacteria used

The volume of bacteria pipetted into a petri dish will remain consistent
regardless of the culture technique it is introduced to.

• Antibiotic used
After the sensitivity test in the pilot study is performed, the type of
antibiotic used (either penicillin or streptomycin) will be confirmed for use
in the experiment itself. This antibiotic will remain consistent in its type
and concentration for every sample it is included in.

Please note
An aseptic technique will be used for the pilot study and the experiment.
Pipettes will be wrapped in foil prior to the trials to increase their sterility,
petri dishes will be kept in a sealed plastic bag and any instrument which is
to be involved in transfers and is less likely to catch fire will be flamed (e.g.
bottle necks, spreaders, forceps).
Outline Method

Three lawn plates were created by first flaming the bottlenecks of the
McCartney bottles containing the nutrient agar. This was then poured into
each of the petri dishes and left to cool and solidify, 0.2cm3 of the culture was
then transferred into each petri dish. The spreader was then dipped in
ethanol and flamed to increase its sterility and left to cool. Once the spreader
had thoroughly cooled, the sample of bacteria in each petri dish was spread
as evenly as possible by revolving each plate slowly on the bench. Forceps
were dipped in ethanol, flamed and left to cool. The antibiotic discs of
penicillin and streptomycin were placed in two of the plates. A disc of sterile
filter paper was placed in the third (to act as a control group).

Three pour plates were created by first flaming the neck of the McCartney
bottle containing the culture, 0.2cm3 of this was transferred into each petri
dish. The bottlenecks of the McCartney bottles containing the nutrient agar
were flamed. These were then poured into each petri dish and left to solidify.
The forceps were then dipped in ethanol and flamed. To increase sterility and
left to thoroughly cool. Then antibiotic discs of streptomycin and penicillin
were added to two of the petri dishes, to the third a sterile filter paper disc
was added as a control.

Equipment

− 6 Sterile Petri dishes, Sterile filter paper discs, Sterile 1cm3 pipettes and
pipette filler

− E.coli in nutrient broth

− Verkon Disinfectant

− Bunsen Burner, Heatproof mat and matches.

− L-shaped spreaders x2, forceps x2, and Antibiotic discs: Streptomycin of

25 µg and Penicillin of 1.5 iu

− Marker Pen, Tape and Scissors

− Ethanol

− Autoclave bag

− Ruler (30cm)

− McCartney bottles filled with nutrient agar.

Accuracy and Precision
(Concerning the Apparatus Used)

Size of the Spreader

• The same sized spreader will be used to create “lawn” plates to ensure
that the amount of growth found on the plates after incubation is
relatively identical for each sample.

Volume of Agar used

• The volume of liquid agar poured into each petri dish will also remain the
same. This is to ensure that the depth of the agar does not affect the
antibiotic action.

The 30cm Ruler

• The ruler will be used to measure the diameter of the inhibition zones but
has an error of 0.5mm. However recording the diameters in whole
millimetres (which incorporate no decimal places) is the most accurate
possible and relatively precise solution in attempting to rectify this error
given the apparatus.

The 1cm3 Pipettes

• The pipettes used have an error of 0.05cm3, and as 0.2cm3 of the bacterial
culture is being transferred this represents a percentage uncertainty of
approximately 25%, which is a relatively high value.
Risk Assessment
Antibiotic discs will only be handled with tweezers to avoid the
possibility of allergic response. Gloves and a lab coat will also be
worn throughout the experiment.
Risk
Contamination of agar plates
Ingestion of bacteria/infection
Contamination of instruments
Precaution
A non-pathogenic strain of e.coli will
be used in this experiment. However
it is also possible to culture
pathogenic bacteria if contamination
occurs, in which case this is
potentially hazardous as the agar is a
nutrient rich medium which would
nourish pathogenic bacteria. To try
and prevent this a Bunsen burner will
be lit on the work surface to ensure
that alien bacteria or fungi from the
environment/air affect the results.
Furthermore when creating a plate,
the lids of dishes are lifted as little as
possible to prevent this. The lids of
the petri dishes were taped and the
necks of containers involved in
transfer were also flamed before and
after use to increase sterility.
As mentioned earlier this strain of
e.coli is non-pathogenic, but hands
will be washed and/or disinfected
before and after the experiment to
prevent culturing of e.coli 0157 (a
pathogen). Hands are also not
permitted to venture anywhere near
the nose or mouth during the
experiment.
The bench will be wiped down with
disinfectant to kill any contaminating
bacteria already present. Gloves will
be worn. Putting instruments down
will be avoided. However if this must
happen, as not to interfere with
experiment, they will be placed near
the Bunsen burner, which will create
an updraft to prevent any bacteria
from falling on the instruments
(which will contaminate the plates).
 The Pilot Study
(A Basic Sensitivity Test)

A basic sensitivity test was done as the pilot study, to gain practice in the
microbiology techniques, discover which antibiotic is the most effective
(creates the larger zones of inhibition) and to also establish where there is
overall room for improvement before the real experiment is executed.

These are the results after 24 hours:

Bacteria Used Culture Antibiotic Units Diameter of

Technique Used ZoI (mm)
used
E.Coli Spread Penicillin 1.5 iu 11
Streptomycin 25 µg 33

E.coli Spread Penicillin 1.5 iu 15

Streptomycin 25 µg 28

E.coli Pour Penicillin 1.5 iu 7

Streptomycin 25 µg 20

E.coli Pour Penicillin 1.5 iu No ZoI ( see *

below)
Streptomycin 25 µg 20

ZoI= Zone of Inhibition

Please note that control groups were made for each culture technique but
could not be included in this table as they were designed to display the
absence of a ZoI.

* In this result the growth of colonies was slightly skewed, as there was no
even distribution of bacteria. Hence there was no growth around the penicillin
disc for it to inhibit.

The results demonstrate that streptomycin is the most effective antibiotic

against this strain of E.coli. Therefore to produce the greatest possible result
in the real experiment streptomycin will be used.
Apparatus

− 12 Sterile Petri dishes, Sterile filter paper discs, Sterile 1cm3

pipettes and pipette filler

− E.coli in nutrient broth

− Verkon Disinfectant

− Bunsen Burner, Heatproof mat and matches.

− L-shaped spreader x2, forceps x2, and Antibiotic discs:

Streptomycin of 25 µg.

− Marker Pen, Tape and Scissors

− Ethanol

− Autoclave bag

− Ruler (30cm)

− McCartney bottles filled with nutrient agar

Method

The investigation was performed in the biology laboratory of Bedford College. All
practical work was supervised and took approximately 1hour and 30 minutes to complete.

All petri dishes were dated and labeled before the experiment.

Preparation of the Spread Plates

1) The bench was wiped down with a disinfectant and a Bunsen Burner was
lit on the surface.

2) A McCartney bottle containing nutrient agar was taken from the water
bath thoroughly dried with a paper towel, to prevent contamination.

3) The lid of the McCartney bottle was removed and the neck of the bottle
was flamed to increase sterility.

4) The lid of the petri dish was opened was opened as little as possible whist
the agar was poured into it.

5) With the plate on the table the agar was gently swirled three or four times
in each of the three directions to ensure even distribution of the agar

6) The steps (2) to (5) were repeated until 3 plates were created.

7) The McCartney bottle of the culture in nutrient broth was shaken well, to
abate any solid cultures that may have been present. The lid was then removed
and the bottleneck was flamed.

8) Once the agar had solidified, 0.2cm3 of the culture was transferred using
aseptic techniques, into a petri dish containing a solid medium of nutrient agar.

9) An L-shaped spreader was dipped in Ethanol, flamed then allowed to burn

and thoroughly cool.

10) The sample of bacteria was spread as evenly as possible by slowly

revolving the plate on the bench.

11) The steps (8) to (9) was repeated for each of the 3 plates.

12) The forceps were dipped in ethanol, flamed then allowed to burn and
thoroughly cool.

13) An antibiotic disc of streptomycin was added to each of the two plates. A
sterile filter paper disc was added to the third to act as a control group.
 (Method cont’d)
Preparation of Pour Plates

1) The bench was swabbed with disinfectant once more.

2) The lid of the McCartney bottle containing the culture in nutrient broth was removed
and the neck was flamed, 0.2cm3 of this was then transferred using aseptic techniques,
into a petri dish.

3) A McCartney bottle containing nutrient agar was removed from the water bath and
dried thoroughly to prevent any contamination.

4) The lid of the bottle was removed and the neck was flamed to increase sterility.

5) The lid of the petri dish was opened as little as possible whilst the nutrient agar was
poured into it.

6) With the plate on the table the nutrient agar was gently swirled three or four times to
ensure an even distribution of agar.

7) The steps (2) to (6) were repeated until 3 plates were created. The agar plates were
then left to solidify.

8) The forceps were dipped in ethanol, flamed and then allowed to burn and thoroughly
cool.

9) A disc of streptomycin was added to each of the two plates. To the third a disc of
sterile filter paper was added, to arise as the control group.

10) The entire experimental procedure was repeated to obtain averages and accuracy.

11) All 12 plates were taped and incubated for 96hours.

12) After incubation, the diameter if the inhibition zones were measured using a 30cm
ruler on the base of the petri dish.

13) Results were then tabulated and all samples were disposed of in an autoclave bag.
 The Experiment
These are the results after 96 hours of incubation.

Please Note: ZoI = Zone of Inhibition, Strep = Streptomycin

Round I

Bacteria Plating Class Diameter Antibioti Units

used Techniqu of ZoI c used
e (mm)
E.coli Spread 4 25mm Strep 25µg

Spread 2 26mm

E.coli Pour 3 23mm Strep 25µg

Pour 1 22mm

Round II

Bacteria Plating Class Diameter Antibioti Units

u Techniqu of ZoI c used
s e (mm)
e
d
E.coli Pour 2 22mm Strep 25µg
Pour 4 22mm

E.coli Spread 3 24mm Strep 25µg

Spread 1 23mm

Table of Averages

Bacteria Plating Diameter Antibiotic Units

Technique of ZoI used
(mm)
E.coli Spread 24.5 Strep 25µg

E.coli Pour 22.5 Strep 25µg

Taking into account all the values produced regardless of which round
they belonged to, the Range (26-22) was found to be 4mm.
The Statistical Test
(Mann Whitney U-Test)

Culture Technique Diameter of ZoI

Spread (NA) 23 24 25 26
RANKA 1 2 3 4
Pour (NB) 22 22 22 23
RANKB 2 2 2 4

ΣRA = 10
ΣRB = 10

NANB= 4 X 4
=16
Analysis

Table of Averages

The average of the results obtained in Round I was taken and then the same
was done for the results obtained in Round II. Hence these two averages were
tabulated in a Table of Averages. The difference between these two averages
is 2mm. This displays some supporting evidence for the hypothesis.

Graph # 1

This is a composite bar chart, which displays the results obtained from Round
I and Round II of the experiment. This chart displays some evidence of
supporting the hypothesis as it proves that spread plating had a slightly
larger effect on the effect of the diameter of the inhibition zone. The Range
(which can be found below the table of results) was calculated to be 4mm,
this displays some variability between the two techniques.

Graph #2

This is a standard bar chart, which only displays the results obtained in Round
I of the experiment. Again this contributes some supporting evidence toward
the hypothesis as the mean difference between the two techniques was
found to be 3mm. This was calculated by finding the difference in the squares
of the bar chart between 1 and 2, and 3 and 4. The mean of these values was
then taken.

Graph #3

This is also a standard bar chart, but it only displays the results obtained in
Round II of the experiment. This also displays supporting evidence towards
the hypothesis. The mean difference was also calculated for this data and
was found to be 1.5mm. This indicates a slight difference between the two
techniques.

There appeared to be no anomalous results for this experiment.

 Analysis (cont’d)

The Statistical Test

The Mann Whitney U-Test was the statistical test used in this experiment. The
U-Test was used to compare the median values between the two sets of data.
It also helped to identify whether there was any difference between them.
The U-Test is a non-parametric test, which allows us to use it when the
sample is relatively small: this is a relevant and important advantage to this
experiment.

After performing the U-test from the results obtained, it can be concluded
that:

The calculated value of u is smaller than the critical value of u for a 1

tailed, n=6 experiment at significance level of 5%. Therefore the null
hypothesis can be rejected (which states that there will be no
significant difference between the ability of the two techniques to
aid the antibiotic’s effectiveness), and we can accept that there is a
correlation between the culture technique used and the diameter of
the zone of inhibition
Conclusion

Collectively observing the results of my statistical analysis and the tabulated

results, they demonstrate some evidence of the effectiveness of the
antibiotic used being affected by the culture technique also. This was
probably due to the fact that one of the techniques provides more oxygen for
aerobic respiration and creates more of a surface area than the other-hence
providing more growth for the antibiotic to inhibit. This technique according
to the results seemed to be spread plating: as in both rounds of the
experiment this technique succeeded in appearing to induce larger diameters
of inhibition-but only by about 2 or 3mm maximum. This subtle difference
could have been due to the bacterial culture used: a non-pathogenic strain of
E.coli. Which as it was mentioned earlier is a versatile and well-adapted
microorganism, which has the ability to grow in the presence or absence of
oxygen.

Further Work

After performing the experiment I feel there is still room for improvement. In
the event of a future investigation, I would:

• Deviate the type of bacterial culture used to establish whether or not it

was the adaptive nature of E.coli that caused subtle differences in the
culture technique and their effect on the diameter of inhibition.

• Make more replicates of both the spread and pour plating technique, as
well as other culturing techniques (e.g. streak plating) to produce a clear
picture of the overall results.

• Investigate the inhibitory effect of metals e.g. coins on bacterial growth

and determine whether this is also affected by culturing techniques

• Alternate the chemical class and concentration of the antibiotic to

determine whether the effect of culture technique is universal for various
antibiotics.

• Perform an antibiotic sensitivity test using more antibiotics (4+) of varied

chemical class and produce samples using different culturing techniques.