BIOL 2P98 2017FW Lab 4

LABORATORY 4

Growth and Identification of Intestinal Bacteria

(Dano, J., 2012; modified by Carpenter-Cleland, C., 2017)

One of the most interesting experiences in an introductory microbiology course is to attempt to identify
an unknown microorganism, and in this lab session you will have the opportunity to attempt this time-honored
skill. Prior to the lab, you and your partners will be required to gather information on a number of organisms
(see Table 4.1 at the end of Lab 3). You will then perform a number of physiological and biochemical tests in lab
on an unknown organism and finally you will compare the results of these tests to the table that you prepared
in advance of the lab. Because of time constraints and to increase the number of tests that you will complete,
in this lab session you and your partner will work with another group to complete the laboratory exercises
(group of 4 students).
Although the fundamental objective of this lab session is to be able to identify an unknown organism,
there is another important objective attached to this exercise. That objective is to gain an understanding of the
cultural and physiological characteristics of bacteria. Physiological characteristics will be determined with a
serious of biochemical tests that you will perform on the organism. Although correctly identifying the unknown
is very important, it is just as important that you thoroughly understand the chemistry of the tests you complete
and the interpretation of the results that you observe (Table 4.2 at the end of Lab 3). Success in this lab will
require meticulous techniques, intelligent interpretation and careful record keeping. Your mastery of aseptic
methods in the handling of cultures and the performance of inoculations will show up clearly in your results.

Background on Biochemical and Metabolic Properties of Microbes

Bacteria, just like human beings, possess a specific set of characteristics bacteria can be classified, or
distinguished from one another based on their cell wall composition and their ability to:
grow on different substrates
produce different end products
produce or express a particular enzyme
use oxygen
be motile

The family Enterobacteriaceae contains well known medically important intestinal bacteria. Organisms in this
family are facultatively anaerobic gram-negative rods that are oxidase-negative and ferment glucose with the
production of acid and gas. Found in soil, water, plants and animals, these organisms are of great economic
importance.

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Figure 4.1 Example of an identification flowchart used to select which tests to use to identify an unknown
bacterium.

To be able to study the cultural, morphological and physiological characteristics of a species, it is

essential to obtain a pure culture of the microorganism (refer to Lab 1). This step has been completed for you.

Biochemical and Physiological Tests

CAUTION: TREAT EACH UNKNOWN AS A PATHOGEN! Inform your instructor of any spills or accidents. WASH
AND SANATIZE YOUR HANDS WELL before leaving the lab.

Gram Stain

Remember all staining work must be carried out over staining trays, and all waste must be
disposed of in waste stain bottles in the fume hood.

Preparation of Heat-fixed smear

Purpose:
To prepare a thin film of bacteria spread on a microscope slide before staining the cells.

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To fix an air-dried film to the slide to prevent cells washing off during the staining, and to denature
bacterial enzymes that could digest the cell wall.

Procedure 1a: Heat-fixed smear

1. Write the name of the bacterium at one end of the slide with your Sharpie.
2. Place a tiny amount of distilled water in the center of the slide.
3. Transfer a very tiny amount of your organism from an agar plate into the water drop with your loop.
4. Spread the mixture into a thin film using the loop and let air dry.
5. Pass the slide quickly through a flame three times, smear side up.

Procedure 1b: Gram Stain

1. Flood the slide with crystal violet dye. Allow this to stand for 1 minute. Then wash gently in distilled
water. Drain.
2. Apply the iodine solution for 1 minute. Wash gently with distilled water. Drain. This reagent acts as a
mordant by binding to the primary stain.
3. Decolorize with Gram's decolorizing fluid, adding drop wise on the tilted slide until all free color (violet)
has been removed (20-30 seconds). Decolorization has occurred when the solvent flows colorless from
the slide.
4. Wash the slide with distilled water.
5. Flood the slide with safranin and allow the stain to act for 40-50 seconds.
6. Again, wash the slide with distilled water.
7. Place a cover slip on the slide and observe under oil immersion objective.
8. Record the results for both organisms.
9. Dispose of all used liquid stain into the bottles in the fume hood. Clean your microscope before placing
back in the cupboard.

Figure 4.2 Gram staining. (a) Procedure

(b) Micrograph of gram-stained bacteria
(Tortora et al, pg 70).

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Before you put the microscope away, always lower the stage, clean the objectives, and rotate the turret to
the 4X objective.

Motility

Many bacteria possess flagellum/flagella and are capable of motility. Flagellum staining is a delicate procedure
and will not be attempted in this lab. We will, however use motility medium to determine motility in different
bacteria species. Motility test medium contains beef extract and peptone to support growth, and a low agar
concentration, 0.5%. A tetrazolium salt (TTC) may be included in the medium to make interpretation easier.
In its oxidized state, TTC is colorless and soluble and when reduced it is red. When a non-motile organism is
stabbed into Motility Test medium, growth occurs only along the line of inoculation, but bacteria with flagella
will move away from the stab line and can be detected by the growth they produce radiating outward from the
stab line. (A Photographic Atlas for the Microbiology Laboratory, pg. 82)

Procedure 2: Motility Determination

1. Collect a Motility medium test tube from the bench and using the coloured tape, label it with
your group numbers, the date, the number of the unknown culture, and Motility. Tape is to be
placed on the glass NOT the lid.
2. Using sterile technique and your inoculation needle inoculate your unknown by means of a stab
inoculation.
3. Incubate your culture at the appropriate temperature for 48 hours.
4. Using the results provided in the Photographic Atlas for the Microbiology Laboratory (pg. 82),
observe your test tube for motility.
5. Examine the tube against a dark background and determine the motility of your organism, record
your results.

Complications - Some motile bacteria, especially the larger ones, simply won't cooperate. Aerobic bacteria may
grow only at the surface, giving the illusion that they are non-motile. If the needle is not pulled straight out, it
may make a second track with growth extending from the first track to the second, falsely indication motility.

Cultural Characteristics

Procedure 3: Cultural Characteristics

1. Collect a 100mm Tryptic Soy Agar plate. Label with your group numbers, unknown culture number,
date and TSA.
2. Perform a Quadrant Streak to isolate single colonies.
3. The plate will be incubated for 24 to 48 hours at the appropriate temperature. Return to collect the
descriptive characteristics of a single colony. Use proper terminology to describe the whole colony
shape, edge/margin, surface and colour. Photographic Atlas for the Microbiology Laboratory (pg.
20) and Appendix D on Sakai.

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Aero tolerance

Microorganisms exhibit great diversity in their ability to utilize free oxygen for cellular respiration. Some
organisms require oxygen for their metabolic needs, others are unaffected by its presence, while others are
killed in its presence. As such, microorganisms can be classified into one of five major groups according to their
oxygen requirements:

Obligate Aerobes: require the presence of atmospheric oxygen for growth. They obtain their energy
through aerobic respiration. An example is Pseudomonas.
Microaerophiles: require a low concentration of oxygen (2% to 10%) for growth. They obtain their
energy through aerobic respiration. Some microaerophiles called capnophiles can survive only if carbon
dioxide levels are elevated. A candle jar may be used to grow these organisms.
Facultative Anaerobic: organisms that can grow either in the presence or absence of oxygen, but
generally grow better in its presence. They obtain energy through aerobic respiration if oxygen is
present, but use fermentation or anaerobic respiration if it is absent.
Obligate Anaerobic: Organisms that are inhibited or even killed by the presence of oxygen. Their
sensitivity is not to oxygen, but rather to by-products of oxygen. These organisms lack the enzymes,
superoxide dismutase, peroxidase or catalase that convert these harmful by-products to harmless
compounds. Note, within this group, there are varying degrees of sensitivity to oxygen. A few molecules
of oxygen kill some methanogens while other organisms, such as Clostridium, can usually survive in
oxygen but cannot grow until conditions become anaerobic. They obtain energy through anaerobic
respiration or fermentation. Examples are Bacteroides and Clostridium.
Aerotolerant: These organisms cannot use oxygen to transform energy but are not adversely affected
by its presence. They do not use oxygen as a terminal electron acceptor. They grow better in conditions
where the oxygen tension is lower than normal atmosphere. These bacteria contain superoxide
dismutase but lack catalase. They obtain energy only by fermentation and are known as obligate
fermenters. An example is Streptococcus.

(A Photographic Atlas for the Microbiology Laboratory, pg. 28)

Procedure 4: Determination of Aero tolerance GasPak method

1. Collect two 60 mm Tryptic Soy agar plates and label each with your group numbers, the date, the number
of your unknown organism and one with Gaspak.
2. Using sterile technique, inoculate each plate with your unknown organism by means of loop inoculation.
Take care to try and use the same amount of inoculant on each plate. Remember you should not be able
to see the inoculant on the plate.
3. Place one plate in the GasPak envelope provided at the front bench. Your TA will seal the envelope to
produce an anaerobic environment, once all plates have been collected. Incubate at the required
temperature. Seal the remaining plate with parafilm and all plates will be incubated at the required
temperature.

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Fluid Thioglycollate Medium (FTM) is a standard medium often used for the determination of oxygen
requirements of bacteria. It will support the growth of common, easily grown chemoheterotrophic bacteria, and
the pattern of growth of an inoculated organism will indicate whether it can respire (using O 2) and/or ferment.
The amino acids and glucose in the medium can be respired, but it is important to note that glucose is the
only fermentable energy source in the medium. It also contains sodium thioglycollate, which binds to oxygen,
thus acting as a reducing agent. Also present is a redox potential indicator, resazurin, which will be a light pink
color in the area of higher oxygen.

Collect a tube of FTM medium and label it with your group numbers, the date, culture number and FTM.
Handle the tubes gently to avoid taking in any unwanted oxygen into the media. If the tube of FTM is pink in
the upper 30% it must be boiled for a few minutes to drive off the oxygen. Place the tube in a boiling water
bath for 10 minutes to drive off the dissolved oxygen from the medium. Cool prior to inoculation.

Procedure 5: Determination of Aero tolerance FTM method

1. Aseptically inoculate the appropriately labeled tube with your unknown test organism by means of loop
inoculations.
2. Incubate the tube at the appropriate temperature for 24 48 hours.
3. Compare the growth of your organism in the tube with diagram in Fig 4.3 and determine the oxygen
requirement of your unknown organism.

Figure 4.3 The appearance of growth in FTM inoculated with different organisms. Each dot represents an
individual bacterial colony within the broth or on its surface (Harley J.P. Prescott L.M., pg 64).

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Differential and Selective media and testing

Purpose: to become familiar with the use and function of specialized media for the selection and differentiation
of microorganisms.

Media can be made with components that will allow for the selective growth of certain organisms and can also
be made to help us differentiate between different groups of organisms.
Selective media are designed to suppress the growth of unwanted bacteria and encourage the growth
of the desired microbe.
A medium is considered differential if it allows us to distinguish between different microbes.

Although the range of selective and differential media that are available is diverse, it is important to
understand that most media have three fundamental features that define their function.
Nutritional components: although many ingredients are suitable for the growth of different microorganisms,
different nutritional components can enhance the overall selectivity of the medium.
Inhibitors: Inhibitors are used to make the medium selective. They are designed to exploit weaknesses in
specific groups of organisms and thus prevent their growth, while allowing other organisms to grow.
Substrates: Differentiation and identification of organisms often relies on their abilities to metabolize certain
substrates or perform certain tasks that can be assayed using specific media and reagents.

Selective and differential media

In the middle of your bench you will find several selective and differential plates. There are a number of
Photographic Atlases for the Microbiology Laboratory available in the lab with background information on the
different selective media. Please take some time and refer to these books, if you did not purchase your own
copy.

Procedure 6:
i. Label each plate with your group numbers, date and unknown organism number. Remember; write
labels along the outside margin of the bottom plate (agar side).
ii. Perform a quadrant streak on all 100mm selective media plates as practice on this technique.
iii. Parafilm around the plates.
iv. Using a small amount of your coloured tape, tape all plates from all procedures together for your two
groups, and label the tape with your group numbers and date then place in the container at the end of
the middle bench.

All plates will be incubated at the appropriate temperature for 24 48 hours. Check for growth when you
return to the viewing time posted on Sakai and the Lab door. (A Photographic Atlas for the Microbiology
Laboratory, pgs. 5-18)

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Respiration Test

Indole Test

Sugar-Indole-Motility (SIM) medium determines indole production from tryptophan by tryptophanase

Organisms that contain the enzyme tryptophanase can utilize the tryptophan in animal and milk
proteins to produce pyruvate. This added source of pyruvate allows for energy production in the Krebs cycle.
The end products from tryptophan metabolism are pyruvate, nitrate and indole. The presence of indole can be
visualized with the addition of Kovacs reagent containing dimethylaminobenzaldehyde and HCl dissolved in
amyl alcohol resulting in Rosindole dye.

Procedure 7: Indole Test

1. Collect a pre-inoculated and pre-incubated SIM test tube, from the bench, containing your unknown.
2. Add 3 drops of Kovacs reagent to the test tube. If the reagent turns red, then your test is a positive result
for indole. (A Photographic Atlas for the Microbiology Laboratory, pg. 74)

Carbon Sources and Fermentation

Kliglers Iron Agar

Heterotrophic bacteria often use sugars in fermentation pathways to obtain usable energy. Organic acid,
alcohols and gases accumulate as waste products: waste products vary depending on the bacteria and their
enzyme complement. In some fermentation pathways, only organic acid and/or alcohols are produced. While
in others gases are released in addition to the organic acids and alcohol. Acid production can be detected by
the addition of a pH indicator to the medium. Gas production can be monitored by the physical lifting of the
agar or fissures containing trapped gas. While most bacteria capable of fermentation can utilize glucose, the
utilization of disaccharides, such as lactose and sucrose, by bacteria requires the production of a specific
hydrolytic enzyme. The set of disaccharide-digesting enzymes made by a particular bacterium contributes to the
microbes unique molecular make-up. The principle behind the Kliglers Iron test is to test the ability of an
organism to ferment glucose and lactose and to determine which organisms can produce hydrogen sulfide. The
media includes a peptone, a small concentration of glucose and a larger concentration of lactose, thiosulphate,
ferric ammonium citrate and a pH indicator. Phenol Red is yellow below pH 6.8, pink above pH 7.4, and red in
between. It must be noted, that deamination of the peptone amino acids producing ammonia may also occur.

Procedure 8 Kliglers Iron Agar

1. Collect a KIA slant from your bench and using the coloured tape, label it with your group numbers, the
date, the number of the unknown culture, and KIA.
2. Using sterile technique and your inoculation needle inoculate your unknown by means of a staband-
streak inoculation.
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3. Incubate your culture at the appropriate temperature for 48 hours to test for hydrogen sulfide. Another
test tube will be provided with 24 hours incubation for fermentaiton results.
4. Using the table provided in the Photographic Atlas for the Microbiology Laboratory (pg. 75), interpret
the results for glucose and lactose fermentation, peptone catabolism, gas production, aerobic use of
peptone, and H2S production.

Methyl Red Test

All enteric bacteria catabolize glucose for their energy production; however, the end products of this
process will vary depending on the specific enzymes present in the bacteria. Mixed acid fermenters produce a
mixture of different organic acids: acetic acid, succinic acid, and formic acid. These acids lower the pH of the
medium to < 5. Butanediol fermenters form butanediol, acetoin, and fewer organic acids. The pH of the medium
does not fall as low as during mixed acid fermentation. When the pH indicator methyl red is added to the
medium and the pH is 4.5 and below, the methyl red reagent remains red in colour. This is a positive methyl
red test. At higher pH values (less acid present), the color may be orange or yellow. These colors denote a
negative test result.

Voges-Proskauer Test

This test was designed for organisms that are able to ferment glucose, but quickly convert their acid
products to acetoin and 2,3-butanediol. Adding Barritts reagent to the culture will detect the presence of
acetoin a precursor in the synthesis of 2,3-butanediol.

Procedure 9 Methyl Red (Mixed Acid Fermentation) and Voges-Proskauer Test (Butanediol Fermentation)
1. Collect a test tube containing MR-VP broth that has been pre-inoculated and incubated for 24 hours with
your unknown organism.
2. Collect a dropper of methyl red indicator and a dropper of VP (Barritts) reagent A and VP (Barritts) reagent
B.
3. Using a sterile tip, transfer 2ml of your culture into an empty sterile test tube and label it VP with coloured
tape.
4. Add five drops of the methyl red indicator to the culture remaining in the original tube. Gently swirl the
tube to mix the broth culture and the pH indicator. Remember to hold the lid in place with your finger while
you shake.
5. Read the reaction immediately. A red colour is a positive test for the mixed-acid fermentation pathway. An
orange or yellow colour is a negative result.
6. Use the Photographic Atlas for the Microbiology Laboratory to interpret the results (pg. 82).
7. To the 2ml aliquot of your culture in the VP labeled test tube, separated before completing the methyl red
test, add 5 drops of VP (Barritts) reagent A and gently shake the culture. Remember to hold the lid in place
with your finger while you shake. Immediately add 5 drops of VP (Barritts) reagent B and shake.
8. Re-shake the culture tube every 5 minutes.
9. Examine and record the colour of the culture after 15 to 30 minutes. The appearance of pink or red at the
top of the broth is a positive result. No color change (yellow) is a negative result. (A Photographic Atlas for
the Microbiology Laboratory, pg. 98)

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Citrate Test

In the absence of fermentable glucose or lactose, some microorganisms are capable of using citrate as a
carbon source for their energy. This ability depends on the presence of citrate permease that can transport the
molecules into the cell and enzymatically convert it to pyruvate and CO2. Pyruvate can then be converted to a
variety of products, depending on the pH of the environment. During this reaction the Simmons citrate medium
becomes alkaline the CO2 that is generated combines with sodium and water to form sodium carbonate, an
alkaline product. This change in pH changes the bromthymol blue indicator incorporated into the medium from
green to deep Prussian blue.

Procedure 10 Citrate Utilization Test

1. Collect a Simmons citrate agar slant from the bench and using the coloured tape, label it with your group
numbers, the date, the number of the unknown culture and Citrate.
2. Using sterile technique and your inoculation needle inoculate your unknown by means of a staband-
streak inoculation.
3. Incubate your culture at the appropriate temperature for 48 hours.
4. Following incubation, citrate-positive cultures are identified by the presence of growth on the surface of
the slant, which is accompanied by blue coloration. Citrate-negative cultures will show no growth and
the medium will remain green. A negative un-inoculated control will be available when you return to
collect your results. (A Photographic Atlas for the Microbiology Laboratory, pg. 64)

Hydrolytic (Digestive) Enzymes

Most high-molecularweight substances (foodstuffs such as polysaccharides, lipids and proteins) are
not able to pass through cell membranes unless first being degraded to smaller molecules. Bacteria produce a
number of different extracellular enzymes (exoenzymes) that act on large molecules outside of the cell
breaking them down so that they can then be transported into the cell.

Gelatin hydrolysis

Derived from collagen, gelatin is a protein used in vertebrate connective tissue. Some organisms have
the ability to hydrolyze gelatin with the enzyme gelatinase, providing the organism with amino acids. An
organism that has the enzyme will liquefy the medium indicating a positive result.

Procedure 11: Gelatin hydrolysis

1. Collect a Nutrient gelatin test tube from the bench and using the coloured tape, label it with your group
numbers, the date, the number of the unknown culture and Gelatin.
2. Using sterile technique and your inoculation needle inoculate your unknown by means of a stab
inoculation.
3. Incubate your culture at 25oC for 48 hours.
4. Using the results provided in the Photographic Atlas for the Microbiology Laboratory (pg. 73) and
Appendix D on Sakai, interpret the results for presence of gelatinases. Indicate if liquefaction has
occurred and the pattern of liquefaction (Appendix D on Sakai).
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Urea hydrolysis

Produced by the decarboxylation of select amino acids, urea can be hydrolyzed to ammonia with the enzyme
urease. Organisms that have urease will be able to hydrolyze urea in the medium, producing ammonia, thus
raising the pH of the medium. Phenol red in the medium will turn pink or red when the pH increases to 8.4 or
higher. Below pH 8.4, phenol red is orange or yellow.

Procedure 12: Urea hydrolysis

1. Collect a Urea broth test tube from the bench and using the coloured tape, label it with your group
numbers, the date, the number of the unknown culture and Urea.
2. Using sterile technique and your inoculation loop inoculate your unknown by means of a simple broth
or plate to broth transfer.
3. Incubate your culture at the appropriate temperature for 48 hours.
4. Using the table provided in the Photographic Atlas for the Microbiology Laboratory (pg. 97); interpret
the results for presence of urease.

Multi-test Systems

In an effort to simplify the identification and speciation of bacteria, particularly the Enterobacteriaceae,
and to reduce the amount of prepared media and incubation space needed by clinical labs, a number of self-
contained multi-test systems have been commercially marketed: RapID ONE System, API system, Microbact
system, and the Enterotube system. Some of these multi-test systems have been combined with a computer-
prepared manual to provide identification based on the overall probability of occurrence for each of the
biochemical reactions. In this way, a large number of biochemical tests can economically be performed in a
short period of time, and the results can be accurately interpreted with relative ease and assurance.

The RapID ONE System is a plastic strip of 18 miniaturized reaction cavities used for the identification of
medically important Enterobacteriaceae and other oxidase-negative, gram-negative rods. Bacterial cultures in
suspension media are added to the micro tubes and the strip is incubated for 4 hours at 35-37oC. Color changes
take place in the tubes either during incubation or after addition of reagents. These color changes reveal the
presence or absence of chemical reactions and, thus, a positive or negative result. The results are recorded in
a score report form and calculated to give a microcode that can be manually interpreted or interpreted using
the online service provide by ThermoFisher Scientific.

Watch the video at the provided web address before coming to perform the lab.
http://www.remel.com/promotions/RapID/Video.aspx

Procedure 13: TA Demonstration of Scoring of RapID ONE System Panel

1. While firmly holding the RapID ONE panel on the benchtop, gently peel the label lid off from the right
hand tab to the left.
2. Read and record results of cavities 1 through 14.
3. Add 2 drops of RapID ONE Reagent into each of cavities 15 through 17; read and record results.
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4. Read the result of cavity 18 and record. Then add 2 drops of RapID ONE Spot Indole Reagent to cavity
18; wait 10 seconds to 2 minutes for a colour change then read and record new results.
5. Identify your unknown using the RapID ONE Differential Chart and Panel to be posted on Sakai. Include
the Report Form for your unknown by inserting it into your electronic report (add a figure number and
a title) as part of the Appendix section.

Inoculation of RapID ONE System Panel TA Demonstration

6. The TA will aseptically inoculate a tube of 2ml RapID Inoculation Fluid to a #2 McFarland turbidity
standard with colonies of one unknown from a 60mm TSA plate provided. Vortex before comparing to
the #2 McFarland Standard.
7. Peel back the lid of the panel over the inoculation port by pulling the tab marked Peel to Inoculate up
and to the left.
8. Using a pipette, transfer the entire contents of Inoculation Fluid into the upper right-hand corner of the
panel. Reseal the inoculation port of the panel by pressing the peel-back tab back in place.
9. After adding the test suspension, while keeping the panel level on the benchtop, tilt the panel back away
from the reaction cavities at a 45o angle.
10. While tilted back, gently rock the panel from side to side to evenly distribute the inoculum along the rear
baffles.
11. While maintaining a level, horizontal position on the bench top, slowly tilt the panel forward to the
reaction cavities until the inoculum flows along the baffles into the reaction cavities. All inoculum should
be evacuated from the rear portion of the panel.
12. Return the panel to a level position on the bench. To remove any air trapped in the cavities, gently tap
the panel on the bench top.
13. Using colour tape, label the panel with date and unknown number.

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Data to be filled in during lab and viewing.

Table 4.3 Provide a title
Unknown organism #__________________
Test Observation/Result of test Interpretation/Meaning
Gram Stain Colour: Circle: GRAM + or -

Cell length (m) Not Required

Cell shape & Not required

arrangement
Colonial Whole colony shape:
characteristics Edge/margin: Not required
Surface:
on TSA
Colour:
Growth on MacConkey: Circle: GRAM + or -
selective EMB:
PEA:
medium
Oxygen Growth in FTM:
requirement Growth in GasPak:
Citrate Test
Indole (SIM)
Voges-Proskauer
Test
Methyl Red Test
Glucose
fermentation
(KIA after 24 hours)
Lactose
fermentation
(KIA after 24 hours)
H2S (KIA or SIM after
48 hours)
Urea hydrolysis
Gelatin
hydrolysis
Motility

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For this laboratory report you must submit a formal report. Refer to Appendix J on How to Write a Formal Lab
Report.

Title Page (5 marks)

Descriptive title, name, lab section, date of study, lab partners.

Abstract (10 marks)

250 word maximum. Must be clear and concise! Briefly summarizes the experimental results obtained
and conclusions. It should contain the purpose, results and conclusions.

Introduction (20 marks)

The introduction should provide the reader with the necessary background information to understand
the results and the discussion of the results. This section should include an introduction to the research,
state why it is important, and briefly survey other work in this field of study. These other works must
be properly referenced. Briefly introduce the identification methods (biochemical tests and RapID)
covered in this lab. Normally it would be necessary to provide the reader with background information
on each biochemical test and organism. You can summarize from Tables 4.1 and 4.2 the distinguishing
characteristics of the possible organisms and the types of tests (cite the tables in the text). Include a
completed NEAT AND READABLE Table 4.1 and Table 4.2 in the Appendix.

Materials and Methods (3 marks)

Reference the Lab Manual and state if and which changes were made.

Results (25 marks)

Following the directions in Appendix J of this manual, present your results in a clear and concise manner.
1. The results of the differential staining and biochemical tests (insert Table 4.3 Data Sheet).
2. An identification flowchart (in the style of Figure 4.1) indicating how you determined your
unknown organism (scan hand drawing and insert if needed).
3. Describe the positive results from the RapID ONE and the identification of unknown. Include the
RapID ONE score report form in the Appendix.

Discussion (25 marks)

Re-state in paragraph form the determination for your unknown. In a step by step manner explain
how you reached this conclusion using the biochemical tests comparing others work (References)
and results to yours. Discuss your results from the RapID test and compare to your individual
biochemical tests.

References (7 marks)
Use the references system of (Author, year) within the text and alphabetical listing in the reference
section.
Example for Lab Manual (only to be used to reference the Materials and Methods):
Carpenter-Cleland, C. (2017) BIOL 2P98 D2 2017FW Principles of Microbiology Lab Manual. St.
Catharines, ON: Brock University.
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Appendix (5 marks)
Any raw data or calculations should be in the appendix (Table 4.1, Table 4.2, RapID ONE Report Form,
etc.).

Working with your 3 partners across the bench (each person is responsible for a different organisms on Table 4.1 and 4
biochemical tests on Table 4.2.) Complete the following tables before coming to Lab 4. All sources must be referenced. Bergeys
manual, Photo. Atlas for the Micro. Lab. and your textbook should have most of the information required. Use other sources to

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complete the tables if needed.

Table 4.1 Physiological and biochemical characteristics of different bacteria.

Escherichia coli
DH5 inactive

Enterobacter

marcescens
Citrobacter
aerogenes

mirabilis

Serratia
freundii

Proteus
Risk Group 2
Cell length (m) 2-6
Gram Stain -
Cell shape & Straight
arrangement bacillus/rods:
singly or pairs
Colonial Smooth
characteristics convex grey
moist or
(pigment colour) rough flat dry
dull wrinkled
Preferred 21-37
Temperature
Range (oC)

Oxygen Facultative
requirement anaerobe
(FTM)
Catalase +
*Glucose A
fermentation (24
hr KIA)
Lactose -
fermentation (24
hr KIA)
H2S (48hr KIA) -
Methyl Red Test +
Voges-Proskauer -
Citrate Test -
Indole Test +
Gelatin hydrolysis -
Urea hydrolysis -
Motility v
*For the fermentation test use A for acid and G for gas
Table 4.2 Biochemical Tests used in Lab 4

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Name of Test Purpose/Importance Name of Indicator or + Reaction - Reaction

Medium Reagent Result Result

Differential PEA
/selective

Differential Mac
/selective Conkey

Differential EMB
/selective

Aerotolerance
Gas Pak

Aerotolerance
FTM

Fermentation Kliglers
of glucose & Iron Agar
lactose 24 hr

H2S Kliglers
Iron Agar
48 hr

Motility

Indole Test SIM

Methyl Red

Voges
Proskauer

Citrate Simmonds
utilization Citrate
Gelatin
hydrolysis
Urea hydrolysis

These 2 tables are to be included in the Appendix of your Lab 4.

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BIOL 2P98 2017FW Lab 4

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