Practical Exam-2 – Title: Cultivation & Identification of Unknown Bacteria.

First of all: the logistics. We are going to use Google Docs, so make sure you have a Gmail account.

You are going to work your paper on Google docs and email me the link to your paper: I will need the
link to give you feedback. When you share the link, make sure you chose allow “Edit”, so I can annotate.

Email your copy anytime before due date, as per the schedule: W-May 12 (by midnight).

When you email me the Google doc link , use my lab email: microbiolab.citytech@gmail.com

The Subject of the email must be: Microbio Practical Lab Report 2

Hopefully not but if you have difficulties with Google docs, email me your word report (no pdf, key,
pages) to microbiolab.citytech@gmail.com; when you save your file, name it as follows:
LastNameFirstName_PracticalLabrep2_MicLab_S21

Reports returned after due date may not be graded, or if graded, may not have feedback, or will have very
late feedback, receive a lower grade.

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Note: The following instructions apply when taking the practical lab exam in class. This does not apply to
online practical, but since you are going to write a report, you still need to review the material and get an
idea how the practical exam is taken in lab.

Session 1.

Choose an “Unknown Culture”. It has a mixture of 2 bac species: one gram-positive and the
other is gram-negative. The do the following:

Write down the number of your unknown sample on your notebook. Then proceed to:

a. Gram stain
b. Streak MacConkey Agar Plate to isolate the gram(-)
c. Streak PEA Agar Plate to isolate the gram+
d. Streak Nutrient Agar Plate NA (control)

Session 2.

Observation of Cultures. Take pictures. Fill the tables on page (2 and 3).

1. Describe the colonies (color, shape, fermentation, etc.) seen on:

a. PEA (gram+)
b. MacConkey Agar Plate (gram-neg)
c. Nutrient Agar NA (control)

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 2. For organisms growing on a MacConkey Agar Plate, after describing the colonies,
inoculate the following media: take the “before “ pictures.
a. BA Blood Agar
b. TSI Agar Slant – remember to stab the medium first then inoculate the slant
c. SIM medium – remember this is inoculated by stabbing the medium only
d. Simmon’s Citrate medium – inoculate the slant only
e. MR/VP broth
f. Urea broth
g. Nitrate Broth

3. Perform Gram Stain on colonies from MacConkey culture.

F20 – Your NAME: MacConkey Results Use Yes/No

Your Unknown Sample # Growth on MacConkey

Gram Stain Color
Shape
Gram Status
Cell Wall
Nutrient Agar: Native Pigmentation,
green? yellow? red? creamy? white?
Blood agar
Beta? Alpha? Gamma?
Lactose fermentation (y/n):
On MacConkey (y/n) Color:
SIM-Indole (y/n) (y/n):
SIM Hydrogen Sulfide (y/n) Color:
Methyl-Red MR (y/n):
(y/n) and color Color:
Vogues-Proskauer VP (y/n):
(y/n) and color Color:
Simmons’ Citrate (y/n) (y/n):
Color:
TSI (color) Slant:

Butt:
TSI-Carbon Dioxide (y/n) (y/n):
Heterolactic? Homolactic? (Hm/Hr) Hm/Hr:
TSI-Hydrogen Sulfide (y/n) (y/n)
Color:
Urease Test (y/n (y/n)
Color:
Nitrate Reductase (y/n) (y/n):
A+B:
+Zinc:

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 4. For organisms growing on PEA agar plate, after describing the colonies, choose isolated
colonies and do the following: take the “before” pictures
a. Inoculate BA,
b. Mannitol
c. Inoculate a Bile Esculin Agar Slant – slant only
d. Inoculate Glucose and TSI
e. Catalase test – remember to choose isolated colonies.
5. Perform Gram Stain on colonies from PEA culture

S20 – Your NAME: PEA Results Use Yes/No

Your Unknown Sample # Growth on PEA

Gram Stain Color
Shape
Gram Status
Cell Wall
Nutrient Agar: Native Pigmentation,
green? yellow? red? creamy? white?
Blood agar
Beta? Alpha? Gamma?
Mannitol Growth (y/n) (y/n)
Mannitol fermentation (y/n) Color:
Fermentation:
Glucose (y/n):
Heterolactic? Homolactic? ((Hm/Hr) Color:
(Hm/Hr)
TSI Growth:
Heterolactic? Homolactic? (Hm/Hr) Color:
Carbon Dioxide:
(Hm/Hr):
Bile Esculin (y/n) (y/n):
Color:
Catalase (y/n) Bubbles:

Simmons Citrate (y/n) (y/n):

Color:

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Session 3.

a. Continue filling the two tables above. You will include the 2 tables and your pictures in your
practical report (results Section).

b. Add all the reagents first. Once you record the results, line up the tubes you inoculated from the
MacConkey Agar plate and take a picture of it – make sure you line up the tube so that your
unknown number and the color change, if any, are clearly seen
c. Do the same for the PEA Agar Plate.

Explanations for the tables shown on pages 2 and 3.

-Gram stain: purple: gram positive or pink: gram negative

-Shape: cocci or rod, big rod, small rod

-Cell wall: thick or thin

-MacConkey Lactose fermentation: pink is yes, dark yellow or brown is no.

-Mannitol growth: yes or no

Mannitol fermentation: yellow: yes, pink: no. When there is no growth, you don’t have to mention
fermentation

-SIM Indole: red: yes, yellow: no

-SIM H2S: black: yes, no black: no

-MR-VP: first split the tube in half.

*MR: red: yes, yellow: no. When MR is red, you don’t have to do VP. When MR is yellow, proceed with
VP on another tube

*VP: red: yes, yellow: no.

-Simmon’s: blue: yes, green: no

-TSI: based on the color, you should be able to tell what sugar was fermented. Slant can be red or yellow;
butt can be red or yellow, butt can be black.

TSI CO2: agar pushed upwards: yes, not pushed: no. heterolactic or homolactic?

TSI-H2S: black: yes, no black: no. Note: TSI sometimes requires a 3-day incubation for H2S to be seen
(you may see discrepancy with SIM-H2S).

-Urease: pink: yes, salmon/yellow: no

-Nitrate reductase: red: yes. Yellow: Zinc it, then: yellow = yes, red: no

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-Bile esculin: black: yes

-Catalase: bubbles: yes

-Oxidase: purple: yes

-Native Pigmentation (not from biochemical tests): creamy, red, yellow, green etc….

Instructions for the Practical Report

Title: CULTIVATION & IDENTIFICATION OF UNKNOWN BACTERIA IN CULTURE #

(do not use a different title)

I suggest you write as we go through the practical, especially the Intro and M&M, references. After each
exam session, write down what you did. To help you with methods, I attached a brief description for each
method (see pages 10-14). You can use that for your paper as long as you list this document in references
(list it as Instructions for PR-2, Prof.)

Follow instructions. Anything you omit from instructions will cost you points for your PR-2 grade.

-You can use and quote any information from your previous lab reports (lab rep #1, practical-rep
1) as long as everything makes sense (don't just copy and paste.)

- Write the Practical-2 lab Report, minimum 5 pages, maximum 7 pages (excluding page 1 as title page,
and last page for references, also images), single-spaced, font size 12, page number and return by the due
date, Wednesday 5/12. No report will be accepted if returned after due the date.

Your paper should have title page: page 1 where you list your name, report title, etc.

*Title page: title, number, with your name, etc.

CULTIVATION & IDENTIFICATION OF UNKNOWN BACTERIA IN CULTURE #

Name:
BIO 3302 – Section - Semester
Etc.

Go to next page:

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*Introduction:

-Introduce the topic of bacteria, what they are, types, etc., and explain why it’s important to grow
(cultivate) bacteria and identify unknown bacteria.

-Briefly introduce all the techniques and media used in this practical, gram, selective, differential,
enriched, carbs, TSI, IMViC, UNCO etc. Be brief, do not go in detail here, leave that for Methods
section, for instance, briefly, PEA is medium that allows the growth of gram+ bacs only,…etc.)

-Clearly state the Objective of the practical exercise, something like the following: …use selective media
to separate unknown gram+ and gram-neg bacs and carry out biochemical tests to identify and name these
unknown bacteria…..

*Materials & Methods : Copy from pages 11 to 14, rephrase the sentences.

-List all the materials used, including the number of your unknown bac mixture. Report your Bacterial
sample number here also. Bench materials such as Bunsen burner, loops, trays, racks, etc. Microscope
materials, slides, immersion oil, etc. Gram stain material. Media: PEA, MacConkey, Blood agars.
Biochemical media: TSI, IMViC, Bile Esculin etc. Reagents for biochemical tests such as Kovac's,
bacterial charts etc. See attached, page 7 and your previous lab reports. This semester we will use
molecular technique, so also add: BioInformatics tools (BLAST)

Describe each method used in detail:

-Bacterial smears, Gram Stain, Microscopy, Streaking agar plates (PEA etc.) Plating bacteria on media
such as TSI , etc. Biochemical tests, IMViC, UNCO, how they done… Indole, oxidase, catalase etc…
Charts to identify bacteria: how they work, Blast, etc.

*Results:

-Insert the picture emailed to you.

-Insert the tables from pages 2 & 3 (please type, avoid attaching a hand-written form). To your best
ability, record the results you see in the image: growth (+/-), color change (biochemical tests), native
pigmentation (pigmentation released by bacteria). This semester we will use molecular technique. You
can add BLAST results for the two unknown bacs here.

*Discussion/Conclusion:

Use bacterial results on the tables to identify the unknown microbes. Use bacterial charts and the attached
key file which has all the bacteria and the results of the tests (data interpretation).

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a- Start with Gram-negative (from MacConkey). Through a process of elimination and a bacterial chart,
discuss your results and attempt to name your unknown gram-neg bacteria. Explain how you came to that
identification.

b- After biochemical identification, gmail me the two names and I will email you the DNA sequences
:gmail the names by Monday 5/10.. After you receive the DNA sequences, proceed to the molecular
identification (Blast). Your biochemical and molecular results may or may not agree. Compare
biochemical results with molecular identification. Are they identical? Yes? Great. No? Explain why.

c- For the final identification: go with the molecular results.

d- Use the info you collected in Homework-1. Once you have the name (from molecular results), give
the taxonomy (classification) of the bacteria: their pathogenicity, disease (s) they caused; perhaps you
can also suggest one or a few antibiotic to kill these bacteria (lab #21) and how the antibiotic will kill
them.

-Do the same as above: a, b, c and d, with Gram-positive bacterium (from PEA).

-Continue writing your paper with a word on the efficiency of the methods used to identify these 2
unknown bacteria: contaminations, limitations, tedious, time consuming, etc.

-End by discussing biochemical and molecular results, other alternatives to identify/name bacteria
(immunological, phage typing, etc.).

-In Conclusion: for each unknown bacteria, conclude with the name, gram status, cell wall, shape and
important biochemical characteristics (fermenter or non-fermenter, etc.)

*References. Mandatory: Use in-text citations as you did for practical 1.

You must list all your sources: at least 6, with complete info. Use a mix of primary (research papers)
and secondary sources (books, reviews, lect/lab slides, Wikipedia, online….).

Example:

Example of a paragraph with in-text citations (underlined for distinction purposes).

Alternatively numbers can be used and the same sequence followed in the list of
references.

Microbes play a crucial role in the carbon and nitrogen cycles on Earth. They are also
responsible for methane production, which not only contributes to global warming but
can also be used as an alternative source of energy against climate change (Vidali M.,
2001). They are also essential for food production, including cheese, bread, and yogurt.
Microorganisms found in soil occasionally cause disease in crops, however they can also
be used to control insect pests and weeds. Many microbiologists work with food
companies to maximize production, develop new food products, and ensure the safety of
medicines, food, and drinks being served to the general population (Jay J.M., 2005).

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 References
Jay, J. M. (2005). Modern food microbiology. New York: Springer.

Manero, A (1999). Identification of Enterococcus spp. With a Biochemical Key. Appl

Environ Microbiol, 65(10), 4425-4430.

Or

Microbes play a crucial role in the carbon and nitrogen cycles on Earth. They are also
responsible for methane production, which not only contributes to global warming but
can also be used as an alternative source of energy against climate change (1). They are
also essential for food production, including cheese, bread, and yogurt. Microorganisms
found in soil occasionally cause disease in crops, however they can also be used to
control insect pests and weeds. Many microbiologists work with food companies to
maximize production, develop new food products, and ensure the safety of medicines,
food, and drinks being served to the general population (2).

References

1. Manero, A (1999). Identification of Enterococcus spp. With a Biochemical Key. Appl

Environ Microbiol, 65(10), 4425-4430.

2. Jay, J. M. (2005). Modern food microbiology.

Practical Lab Rep 2: Grading Rubric

Item Points
Page number 2
Author’s Full Name 2
Title Page, number of unknown sample 2
Introduction (address every instruction) 20
The Objective 5
Materials (list everything used) – see example in instructions file 10
Methods (describe all methods) – see example in instructions file 10
Results: table, images, use only gmailed images 15
Discussion/Conclusion (discuss and answer every question) for each of the 20
two microbes:
Biochemical Identification, Molecular, Classification, Pathogenecity
In Conclusion 5
References (complete) 5
Style (easy read) 4

Total 100

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 Summary

-Day 1: MCQ exam, pick a sample, streak selective media, perform a gram stain

-Day 2: Transfer bacteria to various media for biochemical tests, then re-gram-stain

-Day 3: Reveal biochemical tests.

-Day 4: practical lab report due

After writing your report, check the following:

-Does your report have your name? title? Page number?

-Does your report have all the sections: Intro with objective, M&M, Results with table (printed, not hand-
written), Discussion/Conclusion with a conclusion, References?

-Does M&M section have the number of your unknown bacterial sample?

-Are the paragraphs within each section separated?

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