THE UNIVERSITY OF DAR ES SALAAM.

COLLEGE OF NATURAL AND APPLIED SCIENCES.

DEPARTMENT OF MOLECULAR BIOLOGY AND BIOTECHNOLOGY.

PRACTICAL REPORT.

COURSE CODE: MC 130

COURSE NAME: METHODS AND SAFETY IN MICROBIOLOGY.

STUDENT’S REGISTRATION NUMBER: 2020 – 04 – 06606

STUDENT’S NAME: MCHARO, JONATHAN ABINERI

DEGREE PROGRAM: BSc. Microbiology

DATE OF PRACTICAL:

PRACTICAL NUMBER: Practical 5

PRACTICAL SESSION: CULTURING AND ANALYSIS OF ANAEROBIC BACTERIA.

NAME OF INSTRUCTORS: DR. MPINDA C. B

 ABSTRACT
The aim of this experiment was to isolate and culture(grow) anaerobic bacteria from the soil
sample in an oxygen free setting using the candle technique where the inoculated organism were
put in anaerobic jars that were tightly closed with a lit candle inside. The candle’s aim was to use
up all the available oxygen inside the jar and when the candle went off, the oxygen concentration
was assumed to be very low that would allow only anaerobic bacteria to grow. After incubation
time, the organisms grew in small colonies of white and yellow colony. The bacteria were
observed using techniques such as the gram stain proving to be gram negative on both colony
samples and biochemical tests too were performed on the samples of anaerobic bacteria.
 INTRODUCTION
Anaerobic bacteria are the kind of bacteria that don’t require oxygen for their growth. A few
examples are actinomyces, Clostridium, and Fusobacterium. They are of two types, the obligate
anaerobic bacteria which are strict anaerobes and the facultative anaerobic bacteria.

Growth of anaerobic bacteria especially the obligate requires very special culturing techniques
that would diminish oxygen concentration in their setting because to them, even a small amount
of oxygen can be lethal. Special techniques such as the use of anaerobic jars and anaerobic
incubators can be used for their growth since their growth is also slow.

Anaerobic bacteria can be isolated from different environments including the soil. After
successful growth, these bacteria can be identified and analysed through different techniques
such as the gram staining and biochemical tests such as Oxidase test and Catalase test to
distinguish their synthesis abilities.
 METHODOLOGY
MATERIALS USED:
Materials used in the isolation technique were; A weighed soil sample, 3 bottles containing
99ml of sterile distilled water, a micropipette, micropipette tips, sterile water bottle, 3 sterile petri
dishes, a conical flask containing a hot agar solution (nutrient agar), anaerobic jars, a Bunsen
burner and a candle.

Materials used in Gram staining were; Crystal violet stain, Lugol’s Iodine solution, alcohol
and safranin as the reagents, a clean glass slide, a wire loop and the Bunsen burner.

Materials used in the biochemical test included; Oxidase reagent and Hydrogen Peroxide
solution, 2 clean glass slides, a filter paper.

METHODS/PROCEDURES:

DAY 1:
The three bottles containing 99 ml of sterile distilled water or labelled 10-3, 10-5 and 10-7.

0.1 grams of soil sample was weighed and add to the first bottle of distilled water labelled 10-
3

The bottle was tightly capped and shaken slowly so as to mix the solution.

1 ml of the new solution was transferred from the bottle labelled 10-5 using a new pipette.
The bottle was tightly cape and swirled gently until the solution was mixed.

1 ml of this solution in 10-5 bottle was transferred to the 10-7 bottle with a new pipette. The
bottle was closed and shaken to mix the solution.

One empty sterile dish was taken and 100 ml of the solution from the bottle labelled 10-5 was
poured followed by 100 mL sample of solution from 10-7 bottle to the other second plate.
 The third sterile dish was made control by putting 100 ml of sterile distilled water using
micropipette with a new tip.

Nutrient agar was added on the three plates around the Bunsen burner.

The now inoculated plates were then swirled gently clockwise and anti-clockwise and left to
settle.

The plates were then placed in anaerobic jars and a candle was lit and placed inside the jar
and the jar was tightly sealed.

After about a minute the candle went off and the plates were then incubated in an incubator.

DAY 2:
On the next day, the plates were removed from the incubators and the bacteria colonies on
the plates were counted using a marker and the results were recorded.

Gram staining procedure followed in order to identify the bacteria following these common
Procedures.

Two different colonies were studied; a white colony and yellowish colony.

A thin smear was made on a wet mount and the smear was left to dry at room temperature.

After the smear dried, it was heat-fix By passing the slide 3 times over the Bunsen burner
flame.

Crystal Violet stain was put in few drops on the smear and left for a 1 minute.

The stain was washed off with running water and then covered with Lugol’s Iodine for 30
seconds.

Lugol’s Iodine solution was washed of and the slide was shaken to remove excess water
followed by washing using alcohol for 30 seconds

This smear was washed again with running water then Lastly counter stained with safranin
for 1 minute.
 The smell was dried and bloated using a tissue paper

On the dry smear was put an oil drop which was then observed under a microscope at 1000X
magnification.

Biochemical tests were performed on the bacteria for further observations and analysis. The tests
performed were Oxidase test and Catalase test using the following procedures;

Oxidase test:
Using A filter paper, Oxidase reagent was pored in drops on the paper in two separate
portions

Using the edge of a clean glass slide, bacteria of the white colony was picked from its plate
and then smitten on one wet portion of the filter paper followed by a bacterium from a yellow
colony.

Color change was observed in the first 30 seconds.

Catalase Test:
On a clean glass slide was smitten a yellow colony smear on one edge of the slide and a
yellow colony bacterium smitten on the other edge of the glass light.

On both bacteria smears was added a drop of hydrogen peroxide and left to settle as observations
were made.

RESULTS
After Incubation of the prepared samples in media plates, observations were made and the
following data was obtained;

TABLE SHOWING RESULTS OF PRACTICAL OBSERVATIONS.

 PLATE;
10-5 10-7 CONTROL
RESULTS
GROWTH: Yes Growth Yes Growth Yes Growth
CONTAMINATION: Yes No No
COLONY NUMBER: 7 8 8
COLONY COLORS: Yellow and White Yellow Yellow

Table 1.

After the
Figure 1: Culturing Results on plates.

observations, gram staining was performed on the bacteria samples. The bacteria used were from
a yellow colony and white colony and the magnified images of the bacteria on the microscope
were as follows:

BACTERIA FROM YELLOW COLONY:

The bacteria tested for Gram Negative by failing to retain the primary stain (Crystal Violet) but
turn red as a response to the counter staining of safranin as seen under observations of a
microscope.
Figure2: Gram stain image of Yellow Colony bacteria
BACTERIA FROM WHITE COLONY;

The bacteria from the white colony tested Gram Negative, failing to retain the primary stain
instead agreeing to the counter stain by safranin. The bacterial cells were observed as the below
image;

Figure 3: Gram stain image of white colony bacteria.

After gram staining, the bacteria were taken put into biochemical tests and they gave the
following results;

Oxidase Test;

The test tested Oxidase negative for bacteria sample from white sample but tested Oxidase
positive for bacteria sample from yellow colony.

Figure 4: Oxidase Test Results

Catalase Test;

The test tested Catalase positive for both the bacteria samples as they both produced gas bubbles
upon the addition of Hydrogen Peroxide solution.

Figure 5: Catalase test result

 DISCUSSION
The isolation of anaerobic bactetia can be a challenging task in that it requires careful setting of
apparatus during incubation in a way that oxygen won’t be available. Anaerobic isolation can be
performed in many techniques as the one performed with the candle and an anaerobic jar.

In the experiment, the data obtained confirmed growth of microorganisms in all tge required
plates and apparently on the Control plate too, signifying that there was contamination at the
time of pipetting because the control was to not have growth.

ANSWERS:

Serial dilution is a stepwise or sequential dilution that is performed so as to convert a dense

solution to a more viable concentration. This process is a very important process in studying
microorganisms in that it is helps to reduce the density of cells that are to be inoculated in a plate
agar thus making it possible to obtain organisms with an easily counted number of colonies. In
this experiment, the process was also used and proved efficiency because the colonies came in
few easily countable colonies as demonstrated in the above figure.

Apart from the use of the candle in an anaerobic jar to exclude Oxygen, anaerobic bacteria can
also be isolated and cultured by other methods such as; The use of deep agar tubes (Culturing
away from oxygen, and at times oil is overlaid on the agar surface), the use of anaerobic gas pack
systems (Chemical exclusion of O2 by the use of H2 to convert air to H2O in presence of a
catalyst.) And the use of media containing reducing substances (Robertson cooked meat broth).
 CONCLUSION
In general, the growth and culturing of anaerobic bacteria is difficult and requires very close
attention to the apparatus used and the techniques made in order to make sure no oxygen is left
available in the incubatory setting or else the anaerobes will fail to grow, instead it is the aerobes
that will grow.
Proper culturing and easy analysis/identification of these microorganisms require several steps of
vital importance including the serial distillation, gram staining and biochemical tests for
confirmation and distinguishing of the organisms.
 RECOMMENDATIONS

The theory behind this practical needed effective results in that no contamination was to happen
but seems that this failed due to a low level of aseptic technique. In the upcoming practical, when
conducting isolation, inoculation and culturing of microorganisms it is best to apply aseptic
techniques with more care so as to prevent contamination of the samples leading to uncertain
results.

There should be more accurately working instruments so as to reduce errors in the practical most
especially with the microscopes as most are fault working.
 REFERENCES.
 Benson. 2001. Microbiological application lab manual. McGraw-Hill
companies. 8th edition.
 Martino M.J, Parker J. 1997. Brock biology of Microorganisms. Prentice
hall. 14th edition
 Leboffe M. J, Pierce E. B. 2010. Englewood, Colorado. Microbiology
Laboratory Theory & Application. Douglas N. Morton. 3rd Edition