Bio 2 general

Microbiology Lab
Workbook
PROFESSOR ERIKA CALLE-POPPE AND THAI LY

Long Beach City College

EAST CARSON ST, LONG BEACH, CA 90808
 1

Table of Contents
Purpose of labs………………………….....….2
Lab overview…………………………….....….3
Enzyme Lab Description……………….….…4
1-4 Quadrant Plate Streaking………..….…...6
5-14 DNase……………………..…………….12
5-17 Gelatinase………………………………14
5-13 Starch Hydrolysis(Amylase) …...……..16
4-3 Bile Esculin…………………………….…18
5-18 Urea Hydrolysis (Urease)……………...21
5-12 Phenylalanine Deaminase………….....24
5-11 Lysine Decarboxylase………………….25
5-22 Lysine Iron Agar……………………...…28
5-9 Citrate …………………………….………31
5-6 Catalase …………………………….……33
5-8 Nitrate Reduction ………………………..35
5-20 SIM…………………………….…………39
5-2-Oxidative Fermentation………………....43
5-4-MR/VP…………………………….……....46
5-3-Phenol Red…………………………….....49
5-21 Triple Sugar Iron……………………..…51
4-5-MaConkey…………………………….…..55
4-6-Eosin Methylene Blue (EMB) …………..57
4-7-Hektoen agar ………………………….....59
8-13-BGBL………………………………….….62
4-4-Mannitol Salt..……………………….…….67
5-25-Blood Agar……………………………….70
7-3 Kirby Baur………………………………....72
 2

Purpose of Lab Experiments

1) To identify the genus or species

that bacterium belongs to by
testing each bacteria for “abilities”
that are characteristic for each
different species of bacteria.

2) Use plate,tubes,and agar slants to

grow bacteria and test bacteria for
certain “abilities”.
 3

3 General Bacterial Abilities’ Tested

Enzyme Reactions- reactions that are performed by a
specific enzyme. We want to detect whether a bacteria
can produce a specific enzyme to perform a specific
reaction.
Sugar Utilization-sugars are fermented and are used to
produce energy for the cell. We want to test to see if a
bacteria has the ability ferment a specific sugars.
Selection-components in the medium prevent growth of certain
bacteria and allow growth of others. Eg. Bile. We want to test to
see if a bacterium has the ability grow in presence of harsh
components such as bile or salt.
 4

substrate
Enzyme

Enzyme Reactions
The following labs are detecting enzyme reactions that are
performed by a specific enzyme produced by bacteria. We want
to detect whether a bacteria can produce a specific enzyme to
perform a specific reaction.
What is an enzyme?
It’s a protein! That can break and form chemical bonds which is called a
reaction! Each enzyme performs only specific reaction with a specific
substrate.
Bacteria can release these specific enzymes into the environment.
Write your definition of enzyme: _______________________________
__________________________________________________________
What is a substrate?
It is what the enzyme is reacting with or breaking the chemicals bonds on!
The substrate molecule “fits” into the enzyme perfectly in order for the
enzyme to get close enough to breaks or form bonds in the substrate.
Write your definition of substrate:______________________________
__________________________________________________________
 5

The two types of enzyme reactions are:

1) Hydrolysis Reactions- enzyme is breaking down a
repeating structure into simpler pieces.
List all Enzyme Hydrolysis Reaction Labs:

2) Specific Substrate Reaction- an enzyme is binding a specific

molecule and changing it into another molecule.
List all Specific Enzyme Reaction Labs:
 6

Lab 1-4 Quadrant Plate Streaking

In this experiment, we will be transferring mixed populations of bacteria broths on to two
different agar plates using a loop to streak or spread the bacteria.
Broth #1 Mixed Culture Broth #2 Mixed Culture

Serratia marcescens Pseudomonas aeruginosa

and E.coli and in Liquid and Micrococcus luteus
Nutrient Broth and in Liquid Nutrient
Broth

What is Plate Streaking?

Dilutes a sample of bacteria from a mixed population of either the same or different species.
This method was first devised and used by Loeffler and Gaffky in Robert Koch’s laboratory to
serially dilute bacteria over agar surface and obtain well-isolated colonies. This method is used
to isolate specific bacteria from patient’s infections as well in order to isolate the bacteria
causing the patients infection!
 7

Supplies needed per person:

1. Two NA talls
2. Two sterile plates

Procedure:

1. Melt 2 NA talls and pour into 2 sterile plates.

2. When the Agar has solidified, the prepare plates for streaking.
3. Label the bottom of the NA plates “Broth #1” and “Broth #2” using a Sharpie Marker.
a. Broth #1 Mixed Culture:
i. Serratia marcescens D-1
ii. E. coli
b. Broth #2 Mixed Culture:
i. Pseudomonas aeruginosa
ii. Micrococcus luteus
4. Gently re-suspend the bacteria which have settled to the bottom of
broth BEFORE taking your inoculum.
5. Using aseptic technique, remove one loopful of bacterial suspension from the mixed
broth cultures.
6. Quadrant streaking (spread bacteria) procedure (see drawing to follow):

a) Inoculate the plates by spreading the inoculum over a small area near the edge of
the plate. This is Area 1.
 8

b) Apply the loop lightly to the surface of the agar in order to avoid scratching or
gouging the agar surface.
c) Flame the inoculating loop and let it cool for about 5-10 seconds.
d) Make 4 streaks from Area 1 into Area 2.
e) Flame the loop again and allow it to cool.
f) Make 4 streaks from Area 2 into Area 3, as shown.
g) Flame the loop and allow it to cool.
h) Make 4 streaks from Area 3 into Area 4, as shown.
i) Flame the loop again before putting it down.
j) Repeat steps for broth #2.

Lab 4-1 Quadrant Plate Streaking

Purpose:

Bacteria Background (research each bacterial species in broths 1 and 2):

Hypothesis:

List of Media Ingredients:

List of Materials/Reagents:

Incubation Time and Temperature:

 9

1-4 Pre-Lab Questions

1. Why are we using two broths with two different species in each broth?

2. What is the physical/identifying difference between Serratia marcescens and

E.coli? what about Pseudomonas aeruginosa and Micrococcus luteus?

‘
3. Explain how the plate streaking method can be used to study a sample from a
patient infection.

4. What is a colony and how to they form on an agar plate? Why are we looking to
grow these on each plate?

Results:
Broth #1 Plate Total bacterial colonies:

Total number is each type of colony:

 10

Broth # 2 Plate Total bacterial colonies:

Total number is each type of colony:

1-4 Post-Lab Questions

Do your results match your hypothesis? Why or why not?

Were you able to obtain single colonies on each plate and observe the difference between each
bacterial species in each broth?

Explain any errors that occurred in the experiment and what you will do next time to avoid these
errors.
 11

The following lab experiments are testing for Enzyme Hydrolysis in Bacteria
Lab 5-14 DNase
For experiment 5-14, you will inoculate a single line streak of Serratia marcescens and
Enterobacter aerogenes on each side plate medium using a loop.
Purpose:

Bacteria Background (which bacteria produces the enzyme and which does not):

Hypothesis:

List of Media Ingredients:

List of Extra Materials/Reagents:

Incubation Time and Temperature:

Lab 5-14 Pre-Lab Questions
1) Explain why the plate a blue/teal color.

2) Why is DNA needed in the media?

 12

Draw the results with the appropriate bacterial species written. Also draw which bacteria
is producing the enzyme and show what reaction is occurring.

Lab 5-14 Post-Lab Questions

1) What is the substrate for the enzyme? Explain or draw how this is a hydrolysis reaction?

2) Explain the reason for a bacteria with a clearing around it's growth.

3) Explain the reason for a bacteria without a clearing around it's growth.

4) Why can this test be used to identify staphylococcus aureus?

 13

5) Explain any errors in your experiment or causes of contamination.

Lab 5-17 Gelatin Hydrolysis

For experiment 5-17, you will use a needle to inoculate Proteus vulgaris and E.coli by stabbing
into a separate gelatin talls. You will also include one control tube per group without any
bacteria to incubate in the same area as your bacterial tubes.
Purpose:

Bacteria Background (which bacteria produces the enzyme which does not):

Hypothesis:

List of Media Ingredients:

List of Extra Materials/Reagents:

Incubation Time and Temperature:

Lab 5-17 Pre-Lab Questions
1) Why do we inoculate using a needle?

2) What will happen if you incubate your gelatin tubes at 37 C instead of room temp which is
about 25C (77 F)?

3) What are the two reasons why a gelatin control tall is essential?
 14

4) What is the source of nutrients for growth in this medium?

Draw the results with the appropriate bacterial species and control written for each
tube.

Lab 5-17 Post-Lab Questions

1) What is the substrate for the enzyme? Explain or draw how this is a hydrolysis reaction?

2) Explain the reason for the bacteria that liquifies the gelatin.

3) Explain the reason for a bacteria does not liquify the gelatin.

4) If you tested a bacterium that you expected to be positive for this test but your results
show that the gelatin did not liquefy. Explain two likely reasons this could have occurred.
 15

5) Explain any errors in your experiment or causes of contamination.

Lab 5-13 Starch Hydrolysis

For experiment 5-16, you will inoculate a single line streak of E.coli and Bacillus subtillius on
each side plate medium using a loop.
Purpose:

Bacteria Background (which bacteria produces the enzyme which does not):

Hypothesis:

List of Media Ingredients:

List of Extra Materials/Reagents:

Incubation Time and Temperature:

Lab 5-13 Pre-Lab Questions
1) What is starch? What is a glycosidic bond?

2) Explain the difference between the glycosidic bonds in amylose versus amylopectin starch
molecules?
 16

Draw the results after adding iodine with the appropriate bacterial species written.
Also draw which bacteria is producing the enzyme and show what reaction is
occurring.

Lab 5-13 Post-Lab Questions

1) What is the substrate for the enzyme? Explain or draw how this is a hydrolysis reaction?

2) Explain the results for the bacteria inoculated. What are your conclusions about each
bacterium?

3) How does iodine work chemically with starch? Why can’t iodine bind glucose?
 17

4) If you didn’t add iodine would you still be able to detect starch hydrolysis? Why or why not?

5) What is the source of nutrients in this agar? Why are these nutrients needed?

6) Explain any errors in your experiment or causes of contamination.

Lab 4-3 Bile Esculin Hydrolysis

For experiment 4-3, you will inoculate a single line streak of Enterococcus faecalis and E.coli on
each side plate medium using a loop. Please do not add too much liquid from each bacteria
inoculum!
Purpose:

Bacteria Background (which bacteria produces the enzyme which does not, also which
bacteria can grow on this plate):

Hypothesis:

List of Media Ingredients:

List of Extra Materials/Reagents:

Incubation Time and Temperature:

 18

Lab 4-3 Pre-Lab Questions

1) Explain why this medium is both selective and differential.

2) What reaction indicator is used, how does it work and which reaction is it used to detect?

Draw the results with the appropriate bacterial species written. Also draw which bacteria
is producing the enzyme and show what reaction is occurring. Include the reaction
indicator.
 19

Lab 4-3 Post-Lab Questions

1) What is the substrate for the enzyme? Explain or draw how this is a hydrolysis reaction?

2) Can a bacterium grow on this plate but not be able to hydrolyze Esculin? Explain Why or
why not?

3) If bile was not included in this medium what would be the result for E.coli and Enterococcus
faecalis?

4) If Esculin was not included in the medium, what would be the result for a bacterium that is
expected to test positive?

5) What would be the results of the bacterium Staphylococcus epidermis (Gram-positive skin
bacterium)?

6) Explain any errors in your experiment or causes of contamination.

 20

Lab 5-18 Urea Hydrolysis

For experiment 5-18, you will inoculate a single line streak of Klebsiella pneumonia and E.coli
on each side plate medium using a loop.
Purpose:

Bacteria Background (which bacteria produces the enzyme which does not):

Hypothesis:

List of Media Ingredients:

List of Extra Materials/Reagents:

Incubation Time and Temperature:
Lab 5-18 Pre-lab Questions:
1) Draw the Urea hydrlysis reaction:

2) Are the products of the reaction acidic or basic? Circle them above.

3) Explain how the products cause the pH of the medium to go up or down.

 21

Draw the results with the appropriate bacterial species written. Also draw which bacteria
is producing the enzyme and show what reaction is occurring. Include the reaction
indicator.

Lab 5-18 Post-Lab Questions

1) What is the substrate for the enzyme? Explain or draw how this is a hydrolysis reaction?

2) Which bacteria is pink and why?

3) What reaction indictor is used and why?

4) Explain any errors in your experiment or causes of contamination.

 22

Enzyme Reactions
The following labs are detecting enzyme reactions that
are performed by a specific enzyme produced by bacteria.
We want to detect whether a bacteria can produce a
specific enzyme to perform a specific reaction.
The two types of enzyme reactions are:
1) Hydrolysis- enzyme is breaking down a repeating structure
into simpler pieces.
2) Specific Substrate and Products- enzyme is binding a
specific small molecule and changing it into another
molecule.
Specific Enzyme Labs:
-Lab 5-11 Lysine Decarboxylase
-Lab 5-12 Phenylalanine Deaminase
-5-22-Lysine Iron Agar
-5-6 Catalase
-5-9 Citrate
-5-8 Nitrate Reduction
-5-20 SIM
 23

The following lab experiments are testing for specific Enzyme reactions
Lab 5-12 Phenylalanine Deaminase
Inoculate E.coli and Proteus mirabillis on the Phenylalanine agar slant using the inoculating
loop. You must inoculate using a fish tail motion which is moving the loop side to side on the
surface of the agar slant.
Purpose:

Bacteria Background (which bacteria produces the enzyme which does not):

Hypothesis:

List of Media Ingredients:

List of Extra Materials/Reagents:

Incubation Time and Temperature:

Lab 5-12 Pre-Lab Questions
1) Why are we using an agar slant for this experiment?

2) Why do we inoculate the bacteria in a fish tail motion?

 24

Draw the results with the appropriate bacterial species written for each tube.

Draw the Phenylalanine Deaminase reaction:

The substrate molecule had been partially drawn for you.

N-C-C à
 25

Lab 5-12 Post-Lab Questions:

1) What is the substrate for the enzyme? Explain or draw how the reaction occurred.

2) What reaction indicator is used in this experiment and why is it needed?

3) Explain any errors in your experiment or causes of contamination.

Lab 5-11 Lysine Decarboxylase

For experiment 5-11, you will inoculate Proteus vulgaris and Serratia marcescens using a loop
into a liquid medium and then add mineral oil just before inoculation.
Purpose:

Bacteria Background (which bacteria produces the enzyme which does not):

Hypothesis:

List of Media Ingredients:

List of Extra Materials/Reagents:

Incubation Time and Temperature

 26

5-11 Pre-Lab Questions:

1) What enzyme are we detecting in this test? What is substrate for this enzyme?

2) What components are needed in the medium and what oxygen requirement is needed in
order for the enzyme to be active?

3) What is the reaction indicator for this experiment?

Draw the results with the appropriate bacterial species and control written for each
tube.
 27

Draw the Lysine Decarboxylase reaction:

The substrate molecule had been partially drawn for you.

N-C-C à

5-11 Post-lab Questions:

1) What would be the result for each of the bacteria if mineral oil was not added?

2) Explain how the bacteria changed the medium color to yellow.

3) Explain how the bacteria that changed the medium color to purple.

4) What would be the tube color result in this experiment for bacteria that requires oxygen to
survive?

5) Explain any errors in your experiment or causes of contamination.

 28

Lab 5-22 Lysine Iron Agar

For experiment 5-22, you will inoculate Proteus mirabilis, Enterobacter aerogenes and
Citrobacter freundi into 3 separate tubes using a loop to fishtail inoculate on the agar slant
surface and then stab inoculation with each bacteria using a needle. You must also incubate a
control tube with your bacteria.
Purpose:

Bacteria Background (which bacteria produces the enzyme which does not):

Hypothesis:

List of Media Ingredients:

List of Extra Materials/Reagents:

Incubation Time and Temperature:

Lab 5-22 Pre-Lab Questions
1) Why do we need to stab the agar?

2) What three main reactions that we are testing for in this medium?

3) Why is the coenzyme needed in this medium?

 29

4) What oxygen requirements are needed for deamination versus decarboxylation to occur?

5) Why is a negative control needed for this experiment?

Draw the reactions occurring in this experiment.

Indicate color produced with reaction indicators and enzymes and substrates where
appropriate.
Reaction 1:

N-C-C à
Reaction 2:

N-C-C à

Reaction 3:

Sodium Thiosulfate à
 30

Reaction 4:

Glucose à
Draw the results with the appropriate bacterial species and control written for each
tube.

5-22 Post-Lab Questions

1) What are the results for each of the bacterium tested in this medium? Explain why each of
these results occurred?

2) Is a red slant and purple butt result possible? What reactions are occurred for this result?

3) Why should H2S production occur in the butt of the tube?

 31

4)What would be the result for a bacteria that cannot survive without oxygen (obligate aerobe)?

6) If you grew Shigella flexneri on this medium what would be the results and why

7) Explain any errors in your experiment or causes of contamination.

Lab 5-9 Citrate Test

In lab 5-9 citrate test, you will inoculate E. coli and Enterobacter aerogenes on the surface of a
green agar slant using a needle. Using a needle prevents inoculation of too much bacteria
ensuring that citrate is the only carbon source and prevents false positive.
Purpose:

Bacteria Background (which bacteria produces the enzyme which does not):

Hypothesis:

List of Media Ingredients:

List of Extra Materials/Reagents:

Incubation Time and Temperature:
 32

Lab 5-9 Pre-Lab Questions

1) What component in the medium is the pH indicator? Why is it needed?

2) What is the source of nutrients in this medium for a bacterium that do not have the ability to
utilize citrate?

3) How is this experiment inoculated? Why is it important to inoculate this way?

Draw the results with the appropriate bacterial species written for each tube.

Lab 5-9 Post-Lab Questions

1) Explain the results for the bacteria inoculated. What are your conclusions about each
bacterium?

2) What two results indicate that the bacterium is positive for this test?
 33

3) Are the products of citrate utilization alkaline (basic) or acidic? What color does the tube
change in bacterium that can produce these products?

4) What two enzymes does bacterium need to produce in to breakdown citrate? What is the
function of these two enzymes?

5) Explain any errors in your experiment or causes of contamination.

Lab 5-6 Catalase Test

In lab 5-6 catalase you will inoculate Micrococcus luteus and Enterococcus faecalis on the
surface of two separate nutrient agar slants in order to grow the bacteria for testing to catalase
enzyme.
Purpose:

Bacteria Background (which bacteria produces the enzyme which does not):

Hypothesis:

List of Media Ingredients:

List of Extra Materials/Reagents:

Incubation Time and Temperature:

 34

Lab 5-6 Pre-Lab Questions

1) What type of cell respiration causes H2O2 to form in the cell? Why is a catalase enzyme
needed for this type of respiration to occur?

2) Is it possible for a facultative anaerobic bacterium to produce the enzyme catalase? Why or
why not?

Draw the results with the appropriate bacterial species written for each tube. Draw the
catalase reaction near the bacteria that is positive for catalase.

Lab 5-6 Post-Lab Questions

1) Explain one way you can tell if your test bacteria were contaminated.

2) Why do we need to grow the bacteria in an agar slant before adding H2O2?
 35

3) Explain any errors in your experiment or causes of contamination.

Lab 5-8 Nitrate Reduction Test

In lab 5-8 Nitrate test you will be inoculating Pseudomonas aeruginosa, E. coli and Micrococcus
luteus bacteria into three separate liquid medium tubes using a loop. You must also incubate a
control with your bacteria.
Purpose:
Bacteria Background (which bacteria produces the enzyme which does not):

Hypothesis:

List of Media Ingredients:

List of Extra Materials/Reagents:

Incubation Time and Temperature:

Lab 5-8 Pre-Lab Questions

`1) What type of cellular respiration uses NO3 (Nitrate) as a final electron acceptor?

2) What parts of the nitrate reduction pathway does each bacterium shown in the animation
slides in modules, complete?
 36

Draw the results with the appropriate bacterial species written for each tube. Draw the
chemical form of nitrogen and the reduction pathway completed by each bacterium
present in each tube.
 37

5-8 Post-Lab Questions

1) Fill in the table below based on the results.
Bacteria Result after Result After Zinc Interpretation
Reagent A + B (conclusion)
E.coli Red Produces nitrate
reductase only

Pseudomonas
Aeruginosa

Alcaligenes faecalis

Control (no bacteria)

2) If a bacterium is colorless after addition of zinc, what step in the pathway is this bacterium?

3) If a bacterium is red after adding reagent A and B what does this mean?
 38

4) If you forgot to add reagent B what would be your results? Can you trust these results? Why
or why not?

5) A red color after adding sulfanillic acid (Reagent A) and Alpha amine (Reagent B) indicates
what?

6) Explain the purpose of adding zinc? Which two nitrogen chemical forms are present if the
tube is colorless after you add zinc?

7) What nitrogen chemical form is present in the tube that is RED after you add zinc? Is this a
positive or negative for nitrate reduction?

8) Explain any errors in your experiment or causes of contamination.

 39

Lab 5-20 SIM Test

In lab 5-20 SIM test you will be inoculating E. coli and Proteus mirabilis bacteria into two
separate agar tall tubes. You will use a needle to stab the bacteria into the agar all the way to
the bottom of the tube.
Purpose:

Bacteria Background (which bacteria produces the enzyme which does not):

Hypothesis:

List of Media Ingredients:

List of Extra Materials/Reagents:

Incubation Time and Temperature:

Lab 5-20 Pre-Lab Questions
1) Why is large source of amino acids required for this medium?

2) What is the Kovac’s Reagent and why is it added after incubation?

 40

Draw the results with the appropriate bacterial species written for each tube. Indicate
which bacteria produces the tryptophanase enzyme, which bacteria is motile or produces
H2S gas.

Lab 5-20 SIM Post-Lab Questions

1) Explain the results for the bacteria inoculated. What are your conclusions about each
bacterium?

2) What does that appearance of a bacterial “slime layer” on top of the agar indicate?

3) What are the two ways you can tell bacterium is motile for this test?

4) What would be your conclusion for a bacterium that is red after Kovac’s reagent addition
and has black diffuse precipitate? Can you confirm this bacterium is motile? Why or why
 41

5) Explain the results you would see for an indole negative and motile bacteria for this
test. Name a bacterium that would have this result.

6) Explain any errors in your experiment or causes of contamination.

 42

Non-Selective Sugar Fermentation

Labs
In these labs, specific sugars are in the medium
to test if certain bacteria can or cannot ferment
specific sugars. This medium has nutrients that
allows for growth of all bacteria species.
5-2-Oxidative Fermentation

5-4-MR/VP

5-3-Phenol Red

5-21 Triple Sugar Iron

What are the sugar or sugar(s) that are in each medium?

What are the pH indicators in each medium?

 43

The following lab experiments are testing for fermentation of various sugars in bacteria
without selective media.
Lab 5-2 Oxidative Fermentation Test

In lab 5-2 O/F test you will be inoculating Pseudomonas aeruginosa and E. coli or Enterobacter
aerogenes bacteria into two separate semisolid agar tubes. You will use a loop to stab the
bacteria into the semi-solid agar all the way to the bottom of the tube and add mineral oil just
before incubation. You must also incubate a control with your bacteria.
Purpose:

Bacteria Background (which bacteria ferments specific sugars in the medium):

Hypothesis:

List of Media Ingredients:

List of Extra Materials/Reagents:

Incubation Time and Temperature:

Lab 5-2 Pre-Lab Questions
Define each term.
Facultative anaerobe:

Obligate aerobe:

1) What sugar is in this medium and why is it needed?

 44

2) Why is mineral oil added?

3) Why is this medium a semi-solid medium and not solid or liquid?

Draw the results with the appropriate bacterial species written and control for each tube.
Indicate whether each bacterium completes the oxidative or fermentation pathway. Also,
label which bacteria is a facultative anaerobe and an obligate aerobe.
 45

Lab 5-2 Post-Lab Questions

1) Fill in the table below based on the results.
Bacteria + Mineral Oil Tube No Mineral Oil Glucose Respiration
Result Tube Pathway
Result (Aerobic/oxidative,
Anaerobic/fermentative,
Both or neither)
E.coli or Enterobacter Yellow throughout
aerogenes Tube

Pseudomonas
Aeruginosa

Control (No bacteria)

2) What respiration pathway is occurring for P.aeruginosa in the non-mineral oil tube? How can
you confirm that this is the ONLY pathway that is occurring in this tube?

3) If you forgot to add mineral oil for E.coli can you still confirm that it is a facultative anaerobe?
Why or why not?

4) Why is it important that you stab the bacteria all the way to bottom of the tube? If you did not
stab all the way to the bottom of the tube for E.coli what would be the results?
 46

5) If a bacterium cannot utilize glucose what would be the result in this test? How would this
bacterium grow in the medium? Explain

6) Explain any errors in your experiment or causes of contamination.

Lab 5-4 MR/VP Test

In lab 5-4 MR/VP test you will use a loop to inoculate E. coli and Enterobacter aerogenes
bacteria into two separate liquid medium tubes. You must also incubate a control with your
bacteria.
Purpose:

Bacteria Background (which bacteria ferments specific sugars in the medium):

Hypothesis:

List of Media Ingredients:

List of Extra Materials/Reagents:

Incubation Time and Temperature:

Lab 5-4 Pre-Lab Questions
1) What is the sugar for fermentation in this medium?

2) What is the indicator(s) for the VP and MR tests and why are they needed?
 47

3) Why do you need to do a 5-day incubation for this test?

Draw the results with the appropriate bacterial species written and control for each tube.
Indicate whether each bacterium completes the oxidative or fermentation pathway. Also,
label which bacteria is a facultative anaerobe and an obligate aerobe.

Lab 5-4 Post-Lab Questions

1) Explain the results for the bacteria inoculated. What are your conclusions about each
bacterium?

2) What acid(s) are detected in the MR test versus VP test?

3) Is there a bacterial species that can be positive or negative for both MR and VP tests? If so,
name a bacterium?
 48

4) What would be result for Pseudomonas aeruginosa in this test? Explain this result.

5) Explain any errors in your experiment or causes of contamination.

Lab 5-3 Phenol Red Test
In lab 5-3 phenol red test, you will use a loop to inoculate E. coli, Pseudomonas aeruginosa and
Staphylococcus epidermidis bacteria into separate liquid medium tubes for each sugar (glucose,
lactose and sucrose. You must also incubate a control with your bacteria.
Purpose:

Bacteria Background (which bacteria ferments specific sugars in the medium):

Hypothesis:

List of Media Ingredients:

List of Extra Materials/Reagents:

Incubation Time and Temperature:

5-3 Pre-Lab Questions
1) Is this media selective or differential? Explain why.

2) What pH indicator is in this medium? Why is it needed?

 49

Draw the results with the appropriate bacterial species written and control. Indicate
whether each bacterium is a non-fermenter, fermenter or slow fermenter. Also, label
which bacteria is a facultative anaerobe and an obligate aerobe.
 50

5-3 Post-Lab Questions

Fill in the table of the results based on the results.
Bacteria Tested Sugar fermented Resulting Tube Slow or fast
color fermenter
E.coli Lactose, sucrose,
glucose

1) If the medium turns yellow, what reaction is occurring? Explain.

 51

2) If the medium turns orange, what reaction is occurring? Explain.

3) If the medium turns red/pink, what reaction is occurring? Explain.

4) What extra step can you do to confirm if a bacterium is a non-fermenter result?

5) If a bacterium cannot use the sugar in this medium, can it still grow? Why or why not?

6) Explain any errors in your experiment or causes of contamination.

Lab 5-21 Triple Sugar Iron Test

In lab 5-21 TSI test, you will use a loop to inoculate E. coli, Morganella morganii,Pseudomonas
aeruginosa and Proteus mirabilis bacteria into 4 separate tubes using a loop to fishtail oculate
on the agar slant surface and then stab inoculation with each bacteria using a needle.
Purpose:

Bacteria Background (which bacteria ferments specific sugars in the medium):

Hypothesis:

List of Media Ingredients:

 52

List of Extra Materials/Reagents:

Incubation Time and Temperature:
Lab 5-21 TSI Pre-Lab Questions
1) What component in the medium is the pH indicator? Why is it needed?

2) Why do we need to stab this medium?

3) What two bacterial "abilities" are we testing for in this test?

Draw the results with the appropriate bacterial species written and control. Indicate
which bacterium ferments which sugar and which produces hydrogen sulfide. Also, label
which bacteria is a facultative anaerobe and an obligate aerobe.
 53

Lab 5-21 TSI Post-Lab Questions

Fill in the table below based on the results:
Sugars Fermented
Bacterial Species H2S production (Yes
Color of slant and butt (lactose,sucrose,gl
Tested or No)
ucose)

Escherichia coli

Morganella
morganii

Pseudomonas
aeruginosa

Proteus mirabilis

1) Explain the results for the bacteria inoculated. What are your conclusions about each
bacterium?

2) Why does H2S production only occur in the butt of the tube?

3) What would be results for Staphylococcus epidermidis in this test?

4) What would be the result for Proteus mirabiliis if you forgot to stab the tube during
inoculation?

5) How are glucose only fermenters detected using this test?

6) Explain any errors in your experiment or causes of contamination.

 54

Selective Sugar Fermentation Labs

In these labs, specific sugars in the medium to
test if certain bacteria can or cannot ferment
specific sugars. Medium will contain some
harsh (selective) agent so only specific bacterial
species can grow.
4-5-MaConkey-

4-7 Hektoen Enteric-

8-13-Brilliant Green Bile Lactose (BGBL)-

4-6-Eosin Methylene Blue (EMB)-

4-4-Mannitol Salt-

*List the selective ingredients for each medium*

What are the sugar or sugar(s) that are in each medium?

What are the pH indicators in each medium?

 55

The following lab experiments are testing for fermentation of various sugars in selective
media.
Lab 4-5 MacConkey Agar
For experiment 4-5, you will inoculate a single line streak of Staphylococcus epidermidis,
Salmonella choleraesuis, E. coli or Citrobacter freundi on the plate medium using a loop.
Purpose:

Bacteria Background (which bacteria ferments specific sugars in the medium and can
grow in the medium):

Hypothesis:

List of Media Ingredients:

List of Extra Materials/Reagents:

Incubation Time and Temperature:
4-5 Pre-Lab Questions
Define the two requirements for a bacterium to be considered a Coliform:
1)

2)

1) What pH indicator is in this medium? Why is it needed?

2) Why is this media good for studying only enteric (gut) bacteria?
 56

3) What components in this medium make it differential and selective? Give an explanation for
each.

Draw the results with the appropriate bacterial species written. Indicate which is a
vigorous fermenter of lactose.

4-5 Post-Lab Questions

1) Why can’t Staphylococcus epidermidis grow on this medium?

2) Which bacteria tested is considered a coliform? Explain why.

3) Explain any errors in your experiment or causes of contamination.

 57

Lab 4-6 Eosin Methylene Blue

For experiment 4-6, you will inoculate a single line streak of Enterobacter aerogenes,
Staphylococcus epidermidis and E. coli or Citrobacter freundii on the plate medium using a loop.
Purpose:

Bacteria Background (which bacteria ferments specific sugars in the medium and can
grow in the medium):

Hypothesis:

List of Media Ingredients:

List of Extra Materials/Reagents:

Incubation Time and Temperature:
4-6 Pre-Lab Questions
Define the two requirements for a bacterium to be considered a Coliform:
1)

2)

1) What pH indicator(s) in this medium? Why is it needed

2) Why is this media good for studying only enteric (gut) bacteria?

3) What components in this medium make it differential and selective? Give an explanation for
each.
 58

Draw the results with the appropriate bacterial species written. Indicate which is a
vigorous fermenter of lactose.

4-6 Post-Lab Questions

1) What are the results for each of the bacterium tested in this medium?

2) Can a bacterium still grow on this media if they do not use lactose? Why or Why not?

3) What results would occur for E.coli if lactose was not in the medium?
 59

4) What results would occur for Staphylococcus epidermis if Eosin Y and methylene blue not in
the media?

5) Explain any errors in your experiment or causes of contamination.

Lab 4-7 Hektoen Agar

For experiment 4-7, you will inoculate a single line streak of Shigella flexneri, Salmonella
choleraesuis, E. coli or Citrobacter freundi on the plate medium using a loop.
Purpose:

Bacteria Background (which bacteria ferments specific sugars in the medium and can
grow in the medium):

Hypothesis:

List of Media Ingredients:

List of Extra Materials/Reagents:

Incubation Time and Temperature:
4-7 Pre-Lab Questions
Define the two requirements for a bacterium to be considered a Coliform:
1)

2)

1) What pH indicator(s) in this medium? Why is it needed?

 60

2) What are the fermentable sugars contained in this media?

3) What is the purpose of sodium thiosulfate and iron (III) ammonium citrate in this media?

4) What medium component(s) make this medium differential and selective? Explain how.

Draw the results with the appropriate bacterial species written. Indicate which is a
vigorous fermenter of lactose.
 61

4-7 Post-Lab Questions

Fill in the table below based on the plate results.

Colony/Plate Color H2S Production (Yes Interpretation

Bacteria
results or No) (Conclusions)

Salmonella enterica

Shigella flexneri

Escherichia coli

1) Can different sugars be used in this medium and obtain the same results? Why or why not?

2) Which two bacteria given in the result table would you not be able to distinguish if there was
no sodium thiosulfate present in the media? Why are these bacteria important to
distinguish?

3) Why can salmonella cause a lot of diarrheal sicknesses, but is not considered or defined as
a coliform?

4) Explain any errors in your experiment or causes of contamination.

 62

Lab 8-13 Brilliant Green Lactose Bile Test (BGLB)

In lab 8-13 TSI test, you will use a loop to inoculate E. coli, Morganella morganii,
Staphylococcus epidermidis bacteria into 4 separate tubes using a loop to place bacteria in
liquid media.
Purpose:

Bacteria Background (which bacteria ferments specific sugars in the medium):

Hypothesis:

List of Media Ingredients:

List of Extra Materials/Reagents:

Incubation Time and Temperature:
8-13 Pre-Lab Questions
Define the two requirements for a bacterium to be considered a Coliform:
1)

2)
1) Explain how the bile and dye components make this medium selective? And which bacteria
group of will be resistant the these components and be able to grow.

2) Why can this medium only detect vigorous fermenters of lactose?

3) Why is lactose needed in this medium?

 63

Draw the results with the appropriate bacterial species written and control. Indicate
which bacterium is a coliform and also whether your water sample contains a coliform.

8-13 Post-Lab Questions

1) What are the results for each of the bacterium tested in this medium? Explain why each
of these results occurred?

2) What would be the result for Salmonella enterica in this medium?

3) Can another sugar be used in this medium and you are still able to detect a coliform?

4) Growth and bubble in the durham tube indicates what kind of bacteria has grown?

5) Explain any errors in your experiment or causes of contamination.

 64

Lab 4-7 Hektoen Agar

For experiment 4-7, you will inoculate a single line streak of Shigella flexneri, Salmonella
choleraesuis,E. coli or Citrobacter freundi on the plate medium using a loop.
Purpose:

Bacteria Background (which bacteria ferments specific sugars in the medium and can
grow in the medium):

Hypothesis:

List of Media Ingredients:

List of Extra Materials/Reagents:

Incubation Time and Temperature:

4-7 Pre-Lab Questions

Define the two requirements for a bacterium to be considered a Coliform:
1)

2)

5) What pH indicator(s) in this medium? Why is it needed?

6) What are the fermentable sugars contained in this media?

 65

7) What is the purpose of sodium thiosulfate and iron (III) ammonium citrate in this media?

8) What medium component(s) make this medium differential and selective? Explain how.

Draw the results with the appropriate bacterial species written. Indicate which is a
vigorous fermenter of lactose.
 66

4-7 Post-Lab Questions

Fill in the table below based on the plate results.

Colony/Plate Color H2S Production (Yes Interpretation

Bacteria
results or No) (Conclusions)

Salmonella enterica

Shigella flexneri

Escherichia coli

5) Can different sugars be used in this medium and obtain the same results? Why or why not?

6) Which two bacteria given in the result table would you not be able to distinguish if there was
no sodium thiosulfate present in the media? Why are these bacteria important to
distinguish?

7) Why can salmonella cause a lot of diarrheal sicknesses, but is not considered or defined as
a coliform?

8) Explain any errors in your experiment or causes of contamination.

 67

Lab 4-4 Mannitol Salt Agar

For experiment 4-4, you will inoculate a single line streak of Staphylococcus epidermidis, E.coli
and a sample from your scalp on the salt plate medium and nutrient agar plate medium using a
loop for the bacteria and a cotton swab dipped in sterile sodium chloride to obtain a bacteria
from your scalp.
Purpose:

Bacteria Background (which bacteria ferments specific sugars in the medium and can
grow in the medium):

Hypothesis:
List of Media Ingredients:

List of Extra Materials/Reagents:

Incubation Time and Temperature:

4-4 Pre-Lab Questions
1) What is the pH indicator in this medium? Why is this needed?

2) Why is nutrient agar plate used as a control in this experiment?

3) Why is mannitol needed in this plate?

 68

4) What is the purpose of high salt in this plate?

5) What is the source of nutrients for growth of bacterium that cannot ferment mannitol?

Draw the results with the appropriate bacterial species for the Salt plate and Nutrient
Agar control plate written.

MSA Plate
 69

NA Plate

4-4 MSA Post-Lab Questions

1) Did Staph epidermis grow on this media? Did it turn media yellow? Why or why not?

2) Did Escherichia coli (E.coli) grow on this medium? Why or why not?

3) Is there a non-staph bacterium that can grow and ferment mannitol on this plate? Name
a bacterium. (this must be researched)

4) Explain any errors in your experiment or causes of contamination.

 70

The following lab experiments are diagnostics test used for bacterial infections.
Lab 5-25 Blood agar
You will inoculate your own throat bacteria on to a blood agar plate and swab to test for
presence of Streptococcus pyogenes.
Purpose:

Bacteria Background:

Hypothesis:

List of Media Ingredients:

List of Extra Materials/Reagents:

Incubation Time and Temperature:

5-25 Pre-Lab Questions

1) Why are red blood cells needed in this agar?

2) What is hemolysis? What are the different degrees of hemolysis?

 71

Results:

Post-Lab Questions
1) What is the result of the bacterium that causes strep throat (Streptococcus pyogenes)
for this test? Is your throat sample positive or negative for this bacterium?

2) What is alpha hemolysis and why does it create less clearing in the agar? Name a
bacterium that can perform alpha hemolysis.
 72

Lab 7-3 Kirby Baur

7-3 Lab Questions
1) Why do we need spread bacteria all over the plate?

2) Are you adding the antibiotic disk before or after bacterial growth? Why?

3) Why do we need a comparative standard table of zone of inhibitions?

4) What is the measurement of the zone of inhibition for the antibiotic CIP (ciprofloxacin),
AMP (ampicillin) of the bacteria that you are observing? Is this bacterium resistant or
susceptible to these antibiotics?

Results: Measure the diameter of zone of inhibition and compare to standard chart
Interpretation of Inhibition Zones of Test Cultures

Diameter of Zones of Inhibition (mm)

Disk Disk
symbol Antimicrobial Agent Content
Resistant Intermediate Susceptible

Ampicillin when testing 10 µg

gram-negative bacteria
AM

CIP Ciprofloxacin 5 µg

SXT sulfamethoxazole 23.75 µg

trimethoprim 1.25 µg
TE Tetracycline 30 µg
 73

Interpretation of Inhibition Zones of Test Cultures

Disk Diameter of Zones of Inhibition (mm)
Disk
Symbol Antibiotic Content
Resistant Intermediate Susceptible
Ampicillin when
AM testing 10 µg 16 or less 17 or more
gram-negative
bacteria
Ampicillin when
AM testing 10 µg 28 or less 29 or more
gram-positive
bacteria
B Bacitracin 10 units 8 or less 9 -12 13 or more
CB Carbenicillin when 50 µg 19 or less 18-22 23 or more
testing Proteus
species and E. coli
CB Carbenicillin when 50µg 13 or less 14-16 17 or more
testing P. aeruginosa
C Chloramphenicol 30µg 12 or less 13-17 18 or more
(Chloromycetic)
CIP Ciprofloxacin 5 µg 14 or less 16-20 21 or more
CC Clindamycin 2µg 14 or less 15-20 21 or more
CL Colistin (Coly-mycin) 10µg 8 or less 9-10 11 or more
E Erythromycin 15µg 13 or less 14-22 23 or more
GM Gentamicin 10µg 12 or less 13-14 15 or more
K Kanamycin 30µg 13 or less 14-17 18 or more
ME Methicillin 5µg 9 or less 10-13 14 or more
N Neomycin 30µg 12 or less 13-16 17 or more
NB Novobiocin 30µg 17 or less 18-21 22 or more
OL Oleandomycin 15µg 11 or less 12-16 17 or more
P Penicillin G. when 10 units 28 or less 29 or more
testing staphylococci
P Penicillin G when 10 units 14 or less 22 or more
testing other
microorganisms
PB Polymyxin B 300 units 8 or less 9-11 15 or more
R Rifampin 5µg 16 or less 17-19 20 or more
S Streptomycin 10µg 6 or less 7-9 10 or more
S Sulfonamides 300µg 12 or less 13-16 17 or more
SXT sulfamethoxazole + 23.75 µg 10 or less 13-16 16 or more
trimethoprim 1.25 µg
T(TE) Tetracycline 30µg 14 or less 15-18 19 or more
VA Vancomycin 30µg 9 or less 10-11 12 or more
VA Vancomycin when 30µg 14 or less 15-16 17 or more
testing enterococci