MIDLANDS STATE UNIVERSITY
FACULTY OF SCIENCE AND TECHNOLOGY
NAME : PARDON MAZNHI
REG NUMBER: R184531Z
PROGRAM : APPLIED BIOSCIENCES AND
BIOTECHNOLOGY
MODULE : HABB 202 (BACTERIOLOGY)
LEVEL : 2.1
LECTURER : MISS MGUNI
PRACTICAL WRITEUP TITLE: AGAR PREPARATION, INOCULATION AND
DETERMINANCE OF GROWING MICROORGANISMS.
OBJECTIVES
To show some sources of bacterial contamination and to demonstrate a few techniques available
for evaluating environmental contamination.
To use a differential staining technique to distinguish between gram-positive bacteria and gram
negative bacteria.
To use biochemical reagents and tests for identification of bacteria.
ABSTRACT
The objectives of this experiment were to become familiar with the necessary nutritional and
environmental factors for culturing microorganisms in the laboratory, to understand the
decontamination or sterilization process using an autoclave, to learn the procedures used in
preparing media needed for culturing microorganisms, to learn how to prepare different agar,
inoculate it with bacteria and use various biochemical tests such as the catalase test, oxidase test
and gram staining to identify and classify the bacteria. After the preparation of two different
bacterial media, the plate count agar and nutrient agar, isolation and identification of the different
bacteria was done. The two different types of agar were left to solidify and incubated for 24 hours
at 37oC to allow for bacterial growth. Contamination was minimized by autoclaving the solutions
and materials used for the experiment. After incubation the bacterial colonies that grew were Gram
stain classified and had their Gram status recorded which in this case tested positive to a thick
peptidoglycan. The two bacterial colonies had their colony morphologies studied and also
underwent further biochemical tests, the catalase and oxidase test which are selective media for
further classification.
INTRODUCTION
Bacteria, singular bacterium, are a group of microscopic single-celled organisms that live in
enormous numbers in almost every environment on Earth, from deep-sea vents to deep below
earth’s surface and even to the digestive tracts of humans.
Bacteria lack a membrane-bound nucleus and other internal structures and are therefore ranked
among the unicellular life-forms called prokaryotes (Sadava et al, 2011). Prokaryotes are the
dominant living creatures on Earth, having been present for perhaps three-quarters of Earth
history and having adapted to almost all available ecological habitats. As a group, they display
exceedingly diverse metabolic capabilities and can use almost any organic compound, and some
inorganic compounds, as a food source. Some bacteria can cause diseases in humans, animals, or
plants, but most are harmless and are beneficial ecological agents whose metabolic activities
sustain higher life-forms (Raven and Johnson, 2011). Other bacteria are symbionts of plants and
invertebrates, where they carry out important functions for the host, such as nitrogen fixation and
cellulose degradation. Without prokaryotes, soil would not be fertile, and dead organic material
would decay much more slowly. Some bacteria are widely used in the preparation of foods,
chemicals, and antibiotics. Studies of the relationships between different groups of bacteria
continue to yield new insights into the origin of life on Earth and mechanisms of evolution
(Kramer et al, 1982).
Microorganisms depend on a number of factors such as nutrients, oxygen, moisture and
temperature to grow and divide. In the laboratory, except for the above factors, the culture
medium should be sterile and contamination of a culture with other organisms should be
prevented. A microbiological culture medium must contain available sources of hydrogen donors
and acceptors, carbon, nitrogen, sulphur, phosphorus, inorganic salts and, in certain cases,
vitamins or other growth-promoting substances (Burlage, 1998).
Culture medium or growth medium is a liquid or gel designed to support the growth of
microorganisms. There are different types of media suitable for growing different types of cells.
Nutrient broths and agar plates are the most common growth media, although specialized media
are sometimes required for microorganism and cell culture growth. These agar plates provide a
solid medium on which microbes may be cultured. They remain solid, as very few bacteria are
able to decompose agar. Many microbes can also be grown in liquid cultures comprised of liquid
nutrient media without agar.
A defined medium will have known quantities of all ingredients. For microorganisms, it provides
trace elements and vitamins required by the microbe and especially a defined carbon and
nitrogen source according to Burlage, (1998) . Glucose or glycerol are often used as carbon
sources, and ammonium salts or nitrates as inorganic nitrogen sources. An undefined medium
has some complex ingredients, such as yeast extract, which consists of a mixture of many, many
chemical species in unknown proportions. Undefined media are sometimes chosen based on
price and sometimes by necessity, some microorganisms have never been cultured on defined
media (Sadava et al, 2011).
Since it is necessary to separate bacteria for study, different techniques are applied to do this and
the first step is always to culture the bacteria in a media. There are different types of cultures that
are used and these may include nutritive agar, selective agar and differential agar (Kramer et al,
1982). These types of agar allow different bacteria to grow on them and some agar may allow
growth of only Gram positive bacteria while other media only allows growth of Gram negative
bacteria, and this type of media will be called a differential agar. Nutritive agar provides
nutrition for the growth of almost all types of bacteria. Selective media is a media that allows for
growth of certain bacteria despite their Gram status whether positive or negative (Kramer et al,
1982). When the bacterium has been cultured, it might be separated through biochemical
methods, and this method is used when trying to identify the presence of certain molecules on
the bacterial cell wall or any other cell organelles. Another method that could be used to
differentiate the bacteria is Gram staining, and this method is based on the layer found on some
bacteria called the peptidoglycan. Bacteria that test positive on the Gram test are called Gram
positive bacteria and they have a thick layer of peptidoglycan on its cell wall usually 80% to
95%. Gram negative bacteria have a thin layer of peptidoglycan usually 10% or less in their cell
walls. The Gram positive bacteria will appear purple/violet when viewed under a microscope and
the Gram negative bacteria appears pink in color under microscope (Sadava et al, 2011).
Biochemical tests are the tests used for the identification of bacteria species based on the
differences in the biochemical activities of different bacteria. Bacterial physiology differs from
one species to the other. These differences in carbohydrate metabolism, protein metabolism, fat
metabolism, production of certain enzymes, ability to utilize a particular compound etc. help
them to be identified by the biochemical tests. An example of such biochemical tests are the
oxidase test and the Catalase test. The Catalase Test is used to identify organisms that produce
the enzyme, catalase. This enzyme detoxifies hydrogen peroxide by breaking it down into water
and oxygen gas and the test, if positive results in bubble formation (Sadava et al, 2011).
.
METHODS AND MATERIALS
The materials that were used in this experiment were petri dishes, measuring cylinder, electronic
scale, filter paper, Scott bottles, autoclave, magnetic hot plate, spatula, distilled water, Plate Count
agar, methylated spirit, cotton wool, swabs, milk, sewage water, nutrient agar, loop, slides, cover
slips, microscopes, crystal violet, iodine solution, 95% ethanol, blotting paper, safranin and
incubator. The first step was the preparation of the media which were Plate Count agar and nutrient
agar.
Procedure for the preparation of all the agar solutions
A mass of nutrient agar was weighed using an electric balance. The weighed amount was then
dissolved in 100ml of distilled water. A measuring cylinder was used to measure 100ml and the
solution was poured into a Schott bottle. Using a hot plate, the solution was heated until it reached
boiling point. It was then autoclaved at 121oC for 15 minutes. After it had been autoclaved, the
media was allowed to cool to approximately 37oC and poured into several, clearly labelled with
the type of agar it contained, the date and name of the one who prepared it at their bases. These
were left to solidify. The plates of nutrient agar were exposed to air in the laboratory and the
laboratory corridor for 10 minutes.
A small mass of nutrient agar was weighed using and an electric balance. The weighed amount
was then dissolved in 100ml of distilled water which was measured using a measuring cylinder
and the solution poured in a Schott bottle. Using a hot plate, the solution was heated until it reached
boiling point and the autoclaved at 121oC for 15 minutes. After it had been autoclaved, the media
was allowed to cool to approximately 37oC and poured into several Petri dishes that were clearly
labelled with the type of agar it contained, the date and name of the one who prepared it at their
bases and allowed to solidify. The plates of nutrient agar were exposed to air in the laboratory and
the laboratory corridor for 10minutes. Raw blood was prepared, and diluted with water, whilst
chocolate agar was just heated blood agar.
The plates were all then inverted to prevent the condensed liquid on the lid from dripping into the
growing culture and placed in an incubator at 37oC for 48hours.
Streak Plate
The loop was first flamed to sterilize it and then it was dipped in a beaker containing milk to with
draw some bacteria. The lid of the petri dish containing the solid medium was partially lifted then
the charged loop was held parallel with the surface of the agar and the inoculums were smeared
backwards and forwards across a small area of the medium. The loop was removed and flamed
and then was allowed to cool then the dish was turned through ninety degrees anticlockwise. With
a cooled loop the plate was streaked from area 1 across the surface of the agar in three parallel
lines and small amount of culture was carried over. The loop was removed and the petri dish was
closed. Again the loop was flamed and allowed to cool and the dish also was turned through ninety
degrees anticlockwise. From area two the plate was again streaked across the surface of the agar
in three parallel lines. Furthermore, the loop was removed, flamed and allowed to cool and the
petri dish was closed. Finally, the dish was turned ninety degrees anticlockwise and loop was
streaked across the surface of the agar from area three into the centre of the plate forming zig zag
lines. Lastly the loop was removed and flamed then the petri dish was closed. The plate was then
incubated in an inverted position.
Spread-Plating
A drop of milk was pipetted onto the centre of the plate using a micropipette and the spreader was
sterilized by dipping it into a beaker of ethanol and flaming it. The spreader was cooled by touching
it to the agar near the rim of the plate then the spreader was gently moved back and forth through
the sample across the plate while the turntable is slowly spinning. After closing the lid, the plate
was placed on the bench top undisturbed for five minutes to allow the sample to absorb completely
into the agar. Finally the plate was inverted and incubated.
Catalase Test
Clean slides were taken for each plate, these were sterilized using 70% alcohol, the inoculating
loop was heated until red hot to kill any other bacteria. It was then placed in distilled water and the
water spread on the slide. The loop was heated again and cooled a slightly before contacting the
growing bacteria to avoid killing them. These were spread on a cleaned slide. Drops of hydrogen
peroxide were then added.
Oxidase test
A small piece of filter paper was soaked in 1% Kovacs oxidase reagent and allowed to dry. The
loop was used to pick a well isolated colony from a fresh bacterial plate and then rubbed onto a
treated filter paper. Color changes were observed and recorded.
Gram Staining
A grease free slide was immersed in alcohol. The alcohol was flamed off using a flame in order
to sterilize the slide. The sterilized slide was placed on a clean sheet of tissue and bacteria
samples were collected from the Nutrient agar, blood agar and MacConkey agar in the prepared
plates using an inoculating loop that was flamed until hot then placed in the agarose gel to cool
down. Once it was cooled it was used to scrape the agars in order to extract a single bacterial
colony. The inoculated loop was used to swab the surface of the sterilized slide. The bacteria was
then given some time to dry on to the slide and after was fixed onto the slide by passing the slide
through the flame but ensuring that the bacteria was not cooked and/or killed. The slide was
covered with oxalate-crystal violet for approximately 30 seconds and the slide was rinsed
quickly with a few drops of water. The slide was then wiped on the side without bacteria using
drying paper. The slide was covered with iodine for 2 minutes then rinsed thoroughly with
distilled water. After rinsing, the slide was dabbed with tissue, lightly and without drying, on the
slide where the bacteria were earlier placed. Using 96% ethyl alcohol, the slide was rinsed until
all the colour stains from the dye was washed off. A counter stain was then performed by placing
2.5% alcohol safranin for about 30 seconds. Thereafter, the slide was rinsed with distilled water,
dried on the bottom side of the slide. The results obtained were then recorded
RESULTS
Observations of bacterial growth on the agar used after incubation of 48hrs at 37oC
FIG [Link] AGAR MACCONKY AGAR
BLOOD AGAR
MICROSCOPIC AND MACROSCOPIC RESULTS
Blood Agar
Colony A Colony B
Margin Filiform Undulate
Form Filamentous Irregular
Appearance of the rough smooth
colony surface
Size small large
Colour of the Whitish Greyish colour
colonies
(pigmentation):
Elevation of Flat Flat
bacterial colony
Fig 3.1. Microscopic classification
Colony A Colony B
Gram Status negative negative
Shape Cocci
Fig 3.2 Biochemical Tests
Colony A Colony B
Catalase test Positive Positive
Oxidase Test Positive Negative
Fig 3.3 Macroscopic analysis
MacConky
Colony A Colony B
Margin entire Undulate
Form circular Irregular
Appearance of the
colony surface
Size small small
Colour of the cream pink
colonies
(pigmentation):
Elevation of raised raised
bacterial colony
Fig 3.4. Microscopic classification
Colony A Colony B
Gram Status negative negative
Shape Cocci
Fig 3.5 Biochemical Tests
Colony A Colony B
Catalase test Positive Negative
Oxidase Test Positive positive
Fig 3.6 Macroscopic analysis
Nutrient Agar
Colony A Colony B
Margin undulate curled
Form irregular circular
Appearance of the
colony surface
Size large large
Colour of the cream white
colonies
(pigmentation):
Elevation of Flat Flat
bacterial colony
Fig 3.7. Microscopic classification
Colony A Colony B
Gram Status positive negative
Shape
Fig 3.8 Biochemical Tests
Colony A Colony B
Catalase test negative Positive
Oxidase Test positive
DISCUSSION
From the results obtained it was determined that for bacteria to grow it had to be in a nutritive
medium that supports its growth as well as befitting temperature and pH conditions. Two
nutritive media, blood agar, MacConky and nutrient agar were used for this experiment.
Nutrient Agar is a general purpose, nutrient medium used for the cultivation of microbes
supporting growth of a wide range of non-fastidious organisms, it is non selective which is why
grown bacteria had to go further biochemical tests and the Gram stain test to classify it
(Campbell, 2011).
Bacteria grow tremendously fast when supplied with an abundance of nutrients. Different types
of bacteria will produce different-looking colonies, some colonies may be coloured, some
colonies are circular in shape, and others are irregular (Pressman, 1999). The characteristics of a
colony (shape, size, pigmentation, etc.) are termed the colony morphology. The bacterial species
in this experiment were observed to differ in appearance, texture, shape, number, colour and
elevation, as well as in the three biochemical tests carried out on them. From the obtained
results, major distinctions on colony morphology could be made from the pattern of growth
between the bacterial organisms that grew on the different none selective media. The Gram stain
however in some colonies it tested positive and negative depending on the colony showing that
they had different peptidoglycan in them. In this experiment, we could however not carry out a
colony count because the required tools were malfunctioning at the time the experiment was
carried out (Sadava et al, 2011).
The use of the autoclave’s relevance was shown in this experiment as bacterial samples are very
prone to contamination which could result in biased results or the growth of fungi and yeast
instead of the intended bacteria. According to Burlage, (1998), an autoclave is used to sterilise
solid and liquid media for microbial cultures, heat stable usually the common media ingredients,
heat resistant instruments and equipment, glassware and rubber products. Sterilization in an
autoclave is done with saturated steam under pressure. The high temperatures are responsible for
the killing of microorganisms and not the pressure.
Streaking techniques were used for the isolation of the cultured bacteria. In microbiology,
streaking is a technique used to isolate a pure strain from a single species of microorganism,
often bacteria (Christopher and Bruno 2003). Samples can then be taken from the resulting
colonies and a microbiological culture can be grown on a new plate so that the organism can be
identified, studied, or tested. Some bacteria that grew did not follow the lines of streaking that
had been made but were spread apart throughout the plate and such plate that had this type of
growth was the nutrient agar. The reason for this spread growth in the plates was probably
because the plates had been contaminated. After the culturing of the microorganisms and the
macroscopic analysis which showed that Colony A and colony B had different elevation, color,
size, margin, form and the surface of the colony in each medium, the gram staining was carried
out to further classify the bacteria. Gram positive bacteria stain violet due to the presence of a
thick layer of peptidoglycan in their cell walls, which retains the crystal violet these cells are
stained with. Alternatively, Gram negative bacteria stain pink, which is attributed to a thinner
peptidoglycan wall, which does not retain the crystal violet during the decolouring process, in
this experiment both colonies however tested positive and appeared purple on the smeared slides
(Pressman, 1999).
The biochemical tests done on the bacterial species were the catalase test and the oxidase test.
Catalase is a common enzyme found in nearly all living organisms exposed to oxygen (such as
bacteria, plants, and animals) and catalyses the decomposition of hydrogen peroxide to water and
oxygen and is a very important enzyme in protecting the cell from oxidative damage by reactive
oxygen species. The catalase test demonstrates the presence of catalase, an enzyme that catalyses
the release of oxygen from hydrogen peroxide (H2O2) (Campbell, 2011). It is used to
differentiate those bacteria that produces an enzyme catalase, such as staphylococci, from non-
catalase producing bacteria such as streptococci and for the two bacterial colonies in this
experiment, the test resulted in bubble formation when the hydrogen peroxide was added
meaning they both had catalase.
CONCLUSION
The results showed that both Gram positive and Gram negative bacteria could on nutrient agar,
blood agar and the MacConky and could be isolated by streaking. Sterilization by using an
autoclave is essential to avoid contamination. Colony morphology shows the different growth
patterns and characteristics of bacteria that allow for their classification. Biochemical tests allow
for further classification of bacterial species. Even though there were results, further biochemical
tests such as the indole and urease test could not be carried out because of chemical shortages.
REFERENCES
Burlage, R. S (1998) Techniques in Microbial Ecology. Oxford University Press
Campbell, N.A. (2011) Biology (9thEd), San Francisco: Pearson Benjamin Cummings
Christopher, B. and Bruno, L. (2003) Bacterial colonies and related tests, [Link] (accessed
14/10/2015).
Pressman, R (1999) The Isolation Of Pathogenic Bacteria From the Air, University of
Pennsylvania.
Sadava, D. Hillis, D. Heller, H. and Berenbaum, M. (2011) Life: The science of Biology (9thEd),
Sunderland: Sinauer Associates.
Kramer, J.M. Turnbull, P.C., Munshi, G., and Gilbert, R.J. (1982) Identification and
characterization of Bacillus cereus and other Bacillus species associated with foods and food
poisoning: In Isolation and identification methods for food poisoning organisms: London,
London Academic Press
Mader, S. (2010) Biology(10th ed), New York: McGraw-Hill Companies
Raven, H. and Johnson, G. (2011) Biology(9th ed), New York: McGraw-Hill Companies
Sadava, D. Hillis, D. Heller, H. and Berenbaum, M. (2011) Life: The science of Biology(9th ed),
Sunderland: Sinauer Associates
