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Study Guide for Microbiology (BIO 6) Lab Exam 1
The first laboratory exam will have 50 questions, a combination of short answer and practical
questions. It will be worth a total of 50 points. The practical questions will consist of examples
of lab cultures we grew and observed in our lab exercises as well as microscope specimens
prepared with simple and Gram stains. You will be asked to look at them and answer two or
three questions based on each culture or specimen. The short answer questions will test your
knowledge on the procedures we used, the purpose of each step in the procedures and methods of
interpreting results. YOU WILL BE ALLOWED A 3X5 CARD OF NOTES on this first lab
exam.
Study tips:
The lab study guides are intended to help you focus your attention on the major points covered in
lab. They are not replacements for lab attendance, note taking or completing the required
reading. Any material covered in lab may appear on lab exams, including material not
specifically covered in this study guide.
Where appropriate draw yourself diagrams or flow charts. Flash cards with just one point per
card are also a useful memorization tool.
When you study, I suggest you use this study guide, together with your notes for each week
(both those downloaded from the web and your own notes) and the lab manual. The textbook is
also useful in some instances.
Remember to review the background information, purpose, method, results and interpretation of
results for each experiment. If you need to look up definitions, there is a good glossary at the
back of your lab manual and a good glossary at the back of your textbook.
Exercises included in this exam
Ex. 1-3: Transfer and inoculation technique
Ex. 1-4: Streak Plate Method
Ex. 2-2, 2-3, 2-4: Growth characteristics on plates, slants and broth
Ex. 2-11: Effect of Osmotic Pressure (salt)
Ex. 7-3: Antibiotic Susceptibility (NOT Disinfectants)
Ex. 4-4: Mannitol Salts Agar (as a specialized media that is both selective and differential)
Ex. 4-5: MacConkey Agar
Ex. 5-3: Phenol Red Broth
Ex. 5-5: Catalase Test
Ex. 5-6: Oxidase Test
Ex. 5-14: Casein Hydrolysis Test
Ex. 5-15: Gelatin Hydrolysis Test
Ex. 5-17: Lipid Hydrolysis test
Ex. 5-7: Nitrate Reduction Test
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Week 1 (Ex. 1-3, Ex. 1-4)
Safety Procedures:
What does BSL stand for?
All the organisms we use are classified as BSL-1 and BSL-2. What are the laboratory safety
precautions we use to handle bacteria classified at these levels?
What are the correct procedures for disposing of biohazard waste in the laboratory?
Sterile technique:
In performing sterile or aseptic technique, what techniques are used to prevent crosscontamination of a bacterial culture? How is aseptic technique defined?
Streak plate technique:
How do you perform this technique and what is the purpose? If you do not get the type of growth
you were hoping for, what are possible errors? What is a single colony? What is confluent
growth?
Week 2 (Ex. 2-2, 2-3, 2-4)
Know the terminology used to describe cultural characteristics of plates, broths and slants. Focus
on the terms listed in the Week 2 Lab Notes. Be able to recognize examples of each.
What is the purpose of each type of media (broth, slant, and plate)? What are the advantages and
disadvantages of each culture method?
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Week 3
Antibiotic Sensitivity Experiment (Ex. 7-3)
Explain how you would perform the Kirby Bauer Test. What is meant when we say that this is a
standardized test? What factors are standardized in the Kirby-Bauer test? For this experiment, we
prepared a lawn of bacteria to obtain confluent growth. How would you prepare a lawn? What is
meant by the term confluent growth?
Know how to interpret the results of the Kirby Bauer Test: How do you determine a zone of
inhibition when testing antibiotics? How do you know if the zone of inhibition indicates
resistance or sensitivity? What does it mean for an organism to be resistant to an antibiotic?
Sensitive (same as susceptible)?
How do the results for the Gram positive and Gram negative organisms compare? What is the
most likely reason this difference? Be able to use this information to determine an appropriate
antibiotic to use in a clinical setting.
What is the definition of a narrow spectrum antibiotic? What is the definition of a broad
spectrum antibiotic? What is the advantage and disadvantage of using broad and narrow
spectrum antibiotics in a clinical setting.
Week 4
Effects of osmotic pressure on microbial growth (Ex. 2-11)
Understand the process of osmosis. Know the meaning of the terms hypotonic, isotonic and
hypertonic. If bacteria are placed in a hypotonic, isotonic or hypertonic environment, in each
case, would water move into or out of the cells? What is plasmolysis and when would this
occur?
Define the terms halo- and osmo- and how they relate to the descriptors halophilic, halotolerant,
facultative halophile and obligate halophile.
How do the growth conditions of this experiment relate to the osmotic conditions found in
naturally occurring environments (including inside a cell). Which condition is closest to the
physiological saline concentration of most biological cells?
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Week 5
MICROSCOPY AND STAINING
The Microscope (Ex. 3-1)
Know the parts of the light microscope and the function of each part.
Know how to calculate magnification.
Know the difference between resolution and magnification
Explain focal plane
Explain what are parfocal objective lenses
Explain how immersion oil improves resolution.
Preparation of bacterial smears for microscopy (Ex. 3-5)
How do you prepare a bacterial smear from liquid (broths) and solid (agar) media?
What is the purpose of each step? What does the heat do to the specimen?
Know the differences between a simple stain and a differential stain.
Simple staining (Ex. 3-5)
What are the steps in preparing a simple stain? What information can be obtained about a
bacterial specimen using a simple stain?
Note that the components of a stain are a liquid and a chromogen. The portion of a chromogen
that makes it colored is a chromophore. Chromogens are charged so that they can form ionic or
covalent bonds with components of the cells you wish to stain.
Week 6
Gram stain (Exercise 3-7)
Know the steps in performing a Gram stain, the reagents and the purpose of each reagent
(assume there is a water rinse between each step).
Know how to interpret results (visually identify Gram reactions under a microscope).
Recognize examples of Gram negative and Gram positive organisms.
What is the difference in the structure of the cell walls of Gram-positive versus Gram-negative
bacteria that accounts for the ability of this staining method to differentiate between them? What
is the purpose of Gram staining?
Differential staining (in general)
What is the difference between a simple stain and a differential stain, both in terms of the
information that can be obtained about an organism and in technique? What are the steps
common to performing all the differential stains performed in our lab?
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SOME GENERAL CONCEPTS FOR BIOCHEMICAL TESTS FOR IDENTIFYING
BACTERIA
Biochemical tests allow us to identify when a particular chemical reaction occurs in a cell. In
order to observe the occurrence of a biochemical reaction, some visible change must occur. An
indicator is anything that changes appearance when a biochemical reaction has taken place.
Indicators include color changes, zones of clearing that may form around an area of growth,
bubbling, or any other changes in the growth medium.
Understand what is meant by the term selective medium and what is meant by the term
differential medium. Know whether each of the different media we used were selective,
differential or both. Don't forget that media is used to grow organisms and that all of these tests
tell us something about some aspect of metabolism. The catalase and oxidase tests are not
growth media because the organisms are grown on TSA before the indicator is applied to the
organism.
In a selective medium, an agent is included in the medium that inhibits the growth of unwanted
bacteria and encourages the growth of the species you want to examine. For a selective medium
you need to know what selective agent is included in the medium and what characteristic is
selected for.
Differential media simply change appearance if a particular biochemical reaction takes place
because of the indicators we have already discussed. Differential media contain a substrate that
can be used by an organism only if it produces the enzymes required to catalyze that particular
biochemical reaction. Sometimes the indicator is already present in the medium, sometimes the
indicator may need to be added, and sometimes the indicator is a change in the media itself after
the reaction occurred.
For each biochemical reaction, you need to know:
1) The chemical reaction: substrate  product.
2) The enzyme that catalyzes the reaction.
3) The indicator that is used to determine whether the reaction occurred.
4) How to interpret the change in appearance.
5) The appropriate use for the test.
Since many of these media are based on differences in ability to use various substrates for
fermentation it is important that you understand fermentation reactions (refer to your textbook if
necessary).
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Week 7
SPECIALIZE TESTS (Selective and Differential Growth Media)
1.
Mannitol Salts Agar (Ex. 4-4: MSA - bright red agar plates)
This medium was designed to differentiate between species of Staphylococcus. It is both
selective and differential.
High salt (7.5%) NaCl selects for Staphylococcus. Remember that you learned in your osmotic
pressure experiments that Staphylococcal species are halophiles.
MSA can differentiate between the pathogenic species S. aureus and nonpathogenic species S.
saprophyticus and S. epidermidis, based on the ability to use mannitol as a substrate for
fermentation.
2. MacConkey Agar (Ex. 4-5: dark red agar plate, sometimes brownish red if plate is old)
This medium is designed to easily identify coliform bacteria, specifically E. coli. It is both
selective and differential.
Bile salts and crystal violet inhibit the growth of Gram positive organisms.
This test differentiates between lactose and non-lactose fermenters.
Bile salts and the pH indicator neutral red react with acid fermentation products to produce a
bright pink color.
E. coli is a lactose fermenting organism that produces greater quantities of acid than other lactose
fermenting bacteria. Because of this, it will produce dark pink, magenta colored colonies and
cause bile salt precipitation to produce a bright pink halo in the agar around the area of growth.
Other lactose fermenting bacteria will not react as dramatically and may produce pale pink or red
colonies, but may not turn the agar pink. Pink or red growth is therefore an indication that the
organism is positive for lactose fermentation. Non-lactose fermenting organisms will produce
white colonies and turn the agar a yellowish color.
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3. Phenol Red Broths (Ex. 5-3: red broth in tubes containing an inverted Durham tube)
This medium is used to test for carbohydrate fermentation. This is a differential but not selective
test. This is one of a series of tests that help identify Gram negative rods, although it can be used
with other organisms as well. Expected results are included in the Table in the week 8 Lab
Notes.
If the medium contains glucose, the test will show if an organism is able to use fermentation as a
means of making ATP. The enzymes for glucose fermentation can be generally referred to as
"fermentation enzymes".
What is the pH indicator? What color does it turn if acid fermentation products are present?
What enzyme is present if lactose is used as a substrate for fermentation?
What enzyme is present if sucrose is used for fermentation?
Some organisms produce CO2 and H2 gas in addition to acids as fermentation end products. This
gas will be trapped in the inverted Durham tube if produced.
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Week 8
TESTS TO BE USED ONCE THE GRAM REACTION IS KNOWN
1.
Catalase Activity (Ex. 5-5): Note that this test can be performed on a microscope slide
or directly on an agar medium
This test is used to differentiate Streptococcus from Staphylococcus species.
If hydrogen peroxide (H2O2) is produced in bacteria during electron transfer it is highly toxic
and could kill the cell. Catalase enzymes are used to detoxify a cell of hydrogen peroxide.
To determine if bacteria contain catalase, a drop of hydrogen peroxide is placed on a clean
micoscope slide. A loopful of bacterial culture is smeared into the drop. If it bubbles, O2 is being
released indicating the organism is catalase positive. Even the generation of a small amount of
bubble formation is a positive results. The culture should not be more than 48 hours old.
catalase enzyme
2H2O2 (hydrogen peroxide) ---------------------> 2H2O + O2 (free oxygen gas)
2.
Oxidase Activity (Ex. 5-6: OxiStrips)
This test is used to differentiate enteric (facultative anaerobic) from non-enteric (aerobic)
gram negative bacteria.
Oxidases are enzymes involved in electron transport chain (ETC). Once the electron acceptors
in the ETC have been reduced by NADH or FADH2, they must be reoxidized to be readied for
the next round of electron transport. Cytochrome oxidase is an example of an enzyme that
catalyzes the oxidation these compounds.
We do not have an indicator to look at the steps of the ETC, so in this test we use an artificial
substrate, tetramethyl-p-phenylenediamine dihydrochloride (TPPD), to look for oxidation by
oxidase enzymes because it turns from colorless to purple when oxidized. This substrate is
embedded in the commercially prepared test strips called OxiStrips. Put the test strip in an
empty petri dish and smear a small loopful of bacterial culture on the test strip. It will turn dark
purple within 10 to 15 seconds if an oxidase is present. If a purple color appears after a longer
period of time, the result is negative. It is possible to test several culture samples on the same test
strip. The culture should not be more that 48 hours old.
cytochrome oxidase
reduced cytochrome + O2 ----------------> oxidized cytochrome + H2O
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C. TESTS USED TO IDENTIFY GRAM NEGATIVE NON-ENTERICS
NOTE: Casease, gelatinase and lipase are all exoenzymes. Exoenzymes are enzymes that are
secreted into the surrounding medium and work on substrates found outside the cell. In general,
these exoenzymes are hydrolytic and break down large biomolecules that are too large to be
easily transported into the cell. These biomolecules must be broken down into their smaller
building blocks before they can be made available as a nutrient source for the cell. Starch must
broken down into glucose, protein into amino acids, and triglycerides into fatty acids and
glycerol.
1. Casein Hydolysis Test (Ex. 5-15: milk agar; opaque white agar plates)
These plates contain casein, the major protein found in milk, which gives the plates the milky
color. If an organism produces the exoenzyme casease that digests the protein casein, a zone of
clearing will form around the area of growth.
2. Gelatin Hydrolysis (5-15: gelatin deeps)
Gelatin is produced by boiling collagen (the major protein in bone and connective tissues) and
forms a solid matrix when allowed to cool, much like agar. Organisms that produce the enzyme
gelatinase can hydrolyze the protein into amino acids, which causes it to liquefy. After
incubation of an inoculated tube, test for liquification by cooling the tube in the refrigerator. If
the medium resolidifies, the organism is negative for gelatinase. If the medium remains a liquid,
the organism is positive for gelatinase.
3. Lipase Test (5-17: Tributyrin Agar Plates: opaque, white)
These plates contain the triglyceride tributyrin and test for lipid hydrolysis. The tributyrin makes
the plates an opaque white color. If an organism produces the exoenzyme lipase, a zone of
clearing will form around the area of growth.
4. Nitrate Reduction Test (5-7: Nitrate broth)
This medium is used to assay for the presence of enzymes capable of reducing nitrate to nitrite to
nitrogen gas:
NO3 (nitrate) ----> NO2 (nitrite) ----> N2 (nitrogen gas) or NH3 (ammonia)
What enzyme must be present for conversion of nitrate to nitrite?
The indicators are Nitrate A and Nitrate B reagents. If medium turns red, then nitrate has been
reduced to nitrite.
However, these indicators detect nitrite, so if all nitrite has been further reduced to nitrogen gas
and ammonia, no color would be detected.
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To differentiate between a negative reaction (no reduction and no color change) and complete
reduction to nitrogen gas and ammonia (also no color change due to the absence of NO2), a
small amount of zinc dust is added. Zinc will non-enzymatically reduce any unused substrate,
NO3. Any NO2 produced by the zinc dust will react with the previously added indicator and turn
a red color. This means the bacteria had been unable to reduce nitrate and the result is negative.
If, on the other hand, there is still no color change after adding zinc, then the organism was able
to completely reduced nitrate to nitrogen gas and ammonia.
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