PRACTICAL LAB 4: MICROBIOLOGY

COURSE: FUNDAMENTAL MODULE

COURSE CODE: MDC 201
TITLE OF PRACTICAL: BACTERIA IDENTICATION

INSTRUCTION:
1. Practical lab report needs to be done by INDIVIDUAL student.
2. Each student must answer all the provided questions and draw the diagrams/tests
observed.
3. Mode: Individual.
4. Submission in the form of written report.
5. Date of Submission: 12rd JUNE 2019 (THURSDAY).

PROGRAM LEARNING OUTCOME(S):

PLO1: Apply knowledge of medical sciences in the diagnosis, management and prevention of
common and life-threatening health conditions.
PLO2: Display social responsibility towards patient care with respect to individual and community
health values.

COURSE LEARNING OUTCOME(S):

CLO2: Conduct the various microbiological staining microbiology-slide related diagnostic tools.

TOPIC LEARNING OUTCOME(S):

At the end of this practical session, student should be able to
TLO 1 : Perform biochemical tests on bacterial culture by using given media and reagents

TLO 2: Interpret test results to identify bacteria

INTRODUCTION
In the previous labs you have examined bacteria microscopically. Staining provides valuable
information as to bacterial morphology, gram reaction, and presence of such structures as
capsules and endospores. Beyond that, however, microscopic observation gives little additional
information as to the genus and species of a particular bacterium.

To identify bacteria, we must rely heavily on biochemical testing. The types of biochemical
reactions each organism undergoes act as a "thumbprint" for its identification. Enzymes catalyze
all the various chemical reactions of which the organism is capable. This in turn means that
different species of bacteria must carry out different and unique sets of biochemical reactions.

When identifying a suspected organism, we have to inoculate a series of differential media. After
incubation, observe each medium to see if specific end products of metabolism are present.
This can be done by adding indicators to the medium that react specifically with the end product
being tested, giving some form of visible reaction such as a color change. The results of these
tests on the suspected microorganism are then compared to known results for that organism to
confirm its identification.

In this practical, different bacteria (Gram-positive and Gram-negative) will be demonstrated in

different biochemical reactions due to their unique enzymes. It will also show the results of the
activity of those enzymes. These biochemical tests will be helpful in bacteria identification.
Conduct the following biochemical tests:

1. CATALASE TEST

Procedure:
1. Place a small amount of colony using a clean and dry wooden applicator stick on a glass
slide.
2. Add a few drops of H2O2 to each culture.
3. Observe the evolution of the bubbles.

2. SLIDE COAGULASE TEST

Procedure:
1. Place a small amount of bacterial colony on a glass slide using a wooden applicator stick.
2. Add a drop of plasma on the bacteria colony.
3. Observe for any agglutination within 5 to 10 seconds.

3. OXIDASE TEST

Procedure:
1. Place a drop of 1% tetramethyl-p-phenylenediamine dihydrochloride) on the filter paper.
2. Touch the bacterial colony using an applicator stick on the filter paper.
3. Observe the results within 5-10 seconds.

4. INDOLE TEST

Procedure:
1. Prepare a heavy bacteria suspension in pepton water.
2. Transfer 2-3 drops of bacterial suspension into the indole broth.
3. Incubate the indole broth at 37°C for 24 hours.
4. Add 5 drops of Kovac’s reagent into the inoculated indole broth in the next day.
5. Read the results.
5. METHYL RED (MR) TEST

Procedure:
1. Prepare a heavy bacteria suspension in pepton water.
2. Transfer 2-3 drops of bacterial suspension into the MR broth.
3. Incubate the MR broth at 37°C for 24 hours.
4. Add 5 drops of methyl red into the inoculated MR broth in the next day.
5. Read the results.

6. VOGES-PROSKAUER (VP) TEST

Procedure:
1. Prepare a heavy bacteria suspension in pepton water.
2. Transfer 2-3 drops of bacterial suspension into the VP broth.
3. Incubate the VP broth at 37°C for 24 hours.
4. Add 6 drops of alpha napthol into the inoculated MR broth followed by 2 drops of 40%
KOH in the next day.
5. Shake the VP broth well after the addition.
6. Allow the VP broth to stand for 10-15 min.
7. Read the results immediately.

7. Please examine/observe the provided media and biochemical tests during the practical
session.
RESULTS AND QUESTIONS:

IDENTIFICATION OF GRAM POSITIVE BACTERIA

1. Mannitol Salt Agar (MSA) (Plate provided)

A B

Colony color: Colony color:

Microorganism: Microorganism:

Questions:
a. Is MSA a selective or differential medium? Explain your answer.
______________________________________________________________________
______________________________________________________________________
______________________________________________________________________
______________________________________________________________________

b. Will Streptococcus agalacticae grow on MSA? Explain your answer.

______________________________________________________________________
______________________________________________________________________
______________________________________________________________________
______________________________________________________________________
 2. Blood agar (Plate provided)

A B C

Questions:
a. Draw the hemolysis reactions on A, B and C seen in the provided blood agar.
b. Describe the type of hemolysis by giving the bacteria as example.

Type of hemolysis Descriptions Bacteria

A

C
c. MacConkey (MAC) agar (Plate provided)

A B

Colony color: Colony color:

Fermentation reaction: Fermentation reaction:

Questions:

a. Explain why MacConkey agar is considered as selective and differential medium.

______________________________________________________________________
______________________________________________________________________
______________________________________________________________________
______________________________________________________________________
______________________________________________________________________

b. Name three genus of bacteria that can give fermentation reaction shown in A and B.

A B
4. EMB agar (Plate provided)

Questions:

a. Describe the colony appearance when Escherichia coli growing on EMB.

___________________________________________________________________
___________________________________________________________________

b. Why EMB agar is known as selective and differential medium?

___________________________________________________________________
___________________________________________________________________
___________________________________________________________________
___________________________________________________________________

5. Catalase Test

Positive Negative

Observation: Observation:
Questions:

a. Catalase test has to be conducted with precautions to avoid false positive and false
negative results. List the conditions which can give false positive and false negative
results.

Three false positive:

i. _____________________________________________________________
_____________________________________________________________
ii. _____________________________________________________________
_____________________________________________________________
iii. _____________________________________________________________
_____________________________________________________________

One false negative:

i. _____________________________________________________________
_____________________________________________________________

b. State THREE (3) bacteria that produce catalase positive or catalase negative.

Catalase positive Catalase negative

6. Slide Coagulase Test

Positive Negative

Observation: Observation:

Questions:

a. What reaction does the coagulase enzyme catalyze?

___________________________________________________________________
___________________________________________________________________
___________________________________________________________________

b. Name the bacteria that give coagulase positive and coagulase negative.

Coagulase positive Coagulase negative

IDENTIFICATION OF GRAM NEGATIVE BACTERIA

1. Oxidase Test

Positive Negative

Observation: Observation:

Questions:

a. What is the principle of oxidase test?

______________________________________________________________________
______________________________________________________________________
______________________________________________________________________
______________________________________________________________________

b. Name FOUR (4) genus of bacteria that give oxidase positive and negative in the following
table:
Oxidase positive Oxidase negative
 2. Urease Test

Positive Negative

Observation: Observation:

Questions:

a. What is the principle of urease test?

______________________________________________________________________
______________________________________________________________________
______________________________________________________________________

b. Name THREE (3) genus of bacteria that provide urease positive or negative.

Urease positive Urease negative

3. Indole Test

Positive Negative

Observation: Observation:
 Questions:

a. What is the principle of indole test?

______________________________________________________________________
______________________________________________________________________
______________________________________________________________________
______________________________________________________________________

b. Name ONE indole positive bacteria and THREE indole negative bacteria.

Indole positive Indole negative

4. Methyl Red (MR) Test

Positive Negative

Observation: Observation:

Questions:

a. What is the purpose of MR test?

_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
 b. Name THREE (3) genus of bacteria that provide MR positive or negative.

MR positive MR negative

5. Voges-Proskauer (VP) Test

Positive Negative

Observation: Observation:

Questions:

a. What is the principle of VP test?

_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________

b. Name THREE (3) genus of bacteria that provide VP positive or negative.

VP positive VP negative
6. Citrate Test

Positive Negative

Observation: Observation:

Questions:

a. What is the principle of citrate test?

______________________________________________________________________
______________________________________________________________________
______________________________________________________________________
______________________________________________________________________

b. Name THREE (3) genus of bacteria that provide MR positive or negative.

Citrate positive Citrate negative

7. Triple Sugar Iron (TSI) Agar

Draw the observation:

Escherichia coli Klebsiella sp. Salmonella typhi Proteus sp. Pseudomonas

aeruginosa

Reactions: Reactions: Reactions: Reactions: Reactions:

Slant: Slant: Slant: Slant: Slant:

Butt: Butt: Butt: Butt: Butt:

H2S: H2S: H2S: H2S: H2S:

Gas: Gas: Gas: Gas: Gas:

Symbol for the Symbol for the Symbol for the Symbol for the Symbol for the
reactions: reactions: reactions: reactions: reactions:

Questions:

a. What are the content of TSI agar?

______________________________________________________________________
______________________________________________________________________
______________________________________________________________________
b. How we observe gas production in TSI agar?
______________________________________________________________________
______________________________________________________________________
______________________________________________________________________

c. What is the blackening precipitate indicates in TSI agar? How can it forms in TSI agar?
______________________________________________________________________
______________________________________________________________________
______________________________________________________________________

d. State the types of sugar fermentation can be detected in:

Butt

Slant

8. Motility test

E. coli Klebsiella spp.

Motility Positive Motility Negative

Observation: Observation:
Questions:

a. What is the function of tetrazolium in this test?

_____________________________________________________________________
_____________________________________________________________________

b. How do you tell if the organism is motile based on this test?

_____________________________________________________________________
_____________________________________________________________________

c. Name THREE (3) genus of bacteria that provide motility positive or negative.

Motility positive Motility negative


NO MATRIC NO. STUDENT NAME
Beginning (1)
Participation No preparation for the lab session.
(X2) Show no interest in lab work, no
group discussion occur
Performance Did not follow the correct work
(X2) procedures. No own initiative are
done to ensure the success of the
experiment. Did not perform some
parts of the experiments. Safety
precautions were neglected. Stations
usually in a mess.
Data Illegible writing, loose pages. No
presentation, illustrations. Figures, graphs, tables
Results and contain errors or are poorly
Neatness
constructed, have missing titles,
(X2)
captions or numbers, units missing or
incorrect. The description was not
complete, some parts are missing.
Question Only answer some questions.
answers, Significant problems in grammar,
clarity and spelling or style that make it
accuracy challenging to follow the author's
RUBRIC REPORT
INDIVIDUAL MARKS
Developing (2)
INDIVIDUAL ASSESSMENT
Lack of preparation for the lab session.
Clear on the procedure. inactive
participation in lab, no group discussion
occur
Some work procedures are followed but not
the safety precautions. No own initiative
are done to ensure the success of the
experiment. Performed all parts of the
experiments. Stations are not constantly
neat.
Legible writing, some ill-formed letters,
print too small or too large, papers stapled
together. One illustration. Most figures,
graphs, use tables, some still missing some
important or required features. General
description was provided but not really in
order.
Only answer some questions. Problems in
grammar, spelling or style that interfere
with the author's statements. (Multiple
problems in each paragraph).
Accomplished (3)
Well prepared but need improvement.
Actively participate in lab, group
discussion occur
All the work procedures are followed and
done including the safety precautions. No
own initiative are done to ensure the
success of the experiment. Performed all
parts of the experiments. Stations are
constantly neat.
Legible writing, well-formed characters,
clean and neatly bound in a report cover.
Illustrations provided. All figures, graphs,
tables are correctly drawn, but some have
minor problems or could still be improved.
Description or step-by-step of the process
is included.
Answer all questions. Occasional (2 or 3
per page) grammar, spelling or style
problems. Tendency to use vague words or
excessive jargon. All statements are
Exemplary (4) Score
Well prepared for the lab session.
Very clear on the procedure and
result collection. Actively participate
in lab, group discussion occur, asking
question
All the work procedures are followed
and done including the safety
precautions. Some own initiative are
done to ensure the success of the
experiment. Performed all parts of
the experiments. Stations are
constantly neat.
Word processed or typed, clean and
neatly bound in a report cover.
Illustrations provided creatively. All
figures, graphs, tables are correctly
drawn, are numbered and contain
titles/captions. Description or step-
by-step of the process is included in
detail and in order.
Answer all questions. Grammar,
spelling, and style make it easy for
the reader to follow. Uses words
(X3) statements. More than one inaccuracy
per page. Very incomplete or
incorrect interpretation of trends and
comparison of data. Discussions do
not related with the results obtain
from the experiments. All plagiarism,
copy and paste from internet.
Format, Report done without following the
Timeline given format.
(X1) Report handed in more than one
week late.
Note: The final lab report mark will be converted to 10%.
No more than one minor inaccuracy per
page. Some of the results have been
correctly interpreted and discussed; partial
but incomplete understanding of results is
still evident. Lack of discussion on the data
obtain from experiments.
Report done with some error.
Up to one week late.
accurate relative to the resource being
examined and/or to the material in the
references. Discrepancies are missed.
Almost all of the results have been
correctly interpreted, only minor
improvements are needed. Most results
from the experiment clearly discussed.
Report done by following most of the
format.
Up to two days late.
correctly and avoids jargon unless it
is the most precise word.
All statements are accurate relative to
the resource being examined and/or
to the material in the references. All
important trends and data
comparisons have been interpreted
correctly and discussed, good
understanding of results.
Report done as given format
accurately.
Report handed in on time.
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