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Review for Mid-Term Exam
Microbiology Lab – B. Miller
Note well: This review page covers the main ideas of each of these labs. There may be other
important information in the lab manual, that is NOT covered in this review.
1. Pp. 21-28. Microscopic Examination of Stained Preparations
a. Know parts and functions of microscope.
Part
Function
Stage –
Slide clip –
Coarse adj. –
Fine adj. –
Condenser lens –
Iris diaphragm –
Scanning objective (4X) –
Low Power objective (10X) –
Hi Power objective (40X) –
Oil Immersion objective (100X) –
b. Know technique for viewing very small objects (such as bacteria). That is, one puts bacteria on a
slide, then remarks: “I can’t see anything!”_What steps can you take to make visible that which is
not being seen?
c. Use of the microscope.
i.
What does par focal mean?
ii.
How does having par focal lenses make microscope use easier?
d. Correct steps for cleaning, using, and storing the microscope.
i.
Answer questions about correct (and incorrect) steps for cleaning the microscope.
ii.
Answer questions about correct (and incorrect) steps for using the microscope.
iii.
Answer questions about correct (and incorrect) steps for storing the microscope.
2. Bacterial smears, simple staining, and Gram Stain
a. Pp. 35-38. Preparation of bacterial smears.
i.
Clean slide with detergent. Rinse thoroughly with water and/or alcohol. Allow to dry.
ii.
Hold clean slide by its edge to prevent finger print smudges.
iii.
Label the slide.
iv.
Make a smear from broth culture or solid medium.
A) Add a small drop of water to the slide with the inoculating loop.
B) Heat the loop.
C) Obtain a small sample of a culture (from broth, slant tube, Petri dish)
D) Rub the organisms into the drop of water and smear the mixture over the surface of the
slide.
E) Heat the loop.
v.
Heat fix the organisms to the slide.
A) Allow the smear to dry on the slide… drying may be accelerated by warming the slide
slightly.
B) After the smear is dry, heat the slide slightly by sliding it across the electric incinerator
2/3 times. Do not overheat the slide.
1) Fixation is accomplished when bacterial proteins coagulate and stick to the microscope
slide… like glue.
2) When the bacterial are over-heated, their shapes may be distorted.
b. Pp. 39-42. Simple Stain.
i.
Heat fix the organisms to the slide.
ii.
Common simple stains. One stain only. Apply
A) methylene blue, 1-2 minutes
B) or crystal violet, 20-60 seconds
C) or carbol fuchsin, 15-30 seconds
iii.
Rinse slide with a gently flowing stream of water
iv.
Blot, but do not wipe, the slide with absorbent paper (paper towel).
v.
Observe @ 100X – 1000X
c. Pp. 43-48. *Gram Stain.
i.
Purpose: to distinguish Gram positive bacteria (those with thick peptidoglycan layer in
their cell walls) from Gram negative bacteria (those with thin peptidoglycan layer in their cell
walls). One can also determine shape, size, and arrangement of bacterial cells:
Shape
Coccus (sphere)
Cocci (spheres)
Bacillus (rod)
Bacilli (rods)
Spirilla
ii.
iii.
iv.
Arrangement
Gram-Stain
singles
doubles (diplococcus)
chains (streptococcus)
clusters (staphylococcus)
singles
doubles (diplobacterium)
chains (streptobacterium)
Most cocci are Gm +
Neiserria gonorrhea and
Neiserria meningiditis
are Gm – cocci.
The bacilli are about
50% Gm +
50% Gm -
Singles
Spirilla are Gm -
Size
Cocci (the cells) are the
smallest bacteria
Bacilli are generally
larger than cocci and
smaller than spirilla.
They range from small to
large.
Spirilla are generally the
largest bacteria;
however, some are
nearly as small as large
bacilli.
Make a smear from broth culture or solid medium.
Heat fix the organisms to the slide.
Apply the Gram-stain reagents
A) Apply Primary Stain (Gram I, Crystal Violet): 1-2 minutes.
1) This stain will stain both Gram+ and Gram- cells purple.
2) However, the purple stain will at a later step be washed out of the Gram- bacteria.
3) Rinse slide with water to remove excess crystal violet solution
B) Apply Mordant (Gram II, Iodine): 1 minute
1) The iodine binds to the crystal violet (CV), forming an insoluble I-CV complex, which
attaches to the peptidoglycan in Gram+ cell walls.
2) All cells will appear dark purple at this stage.
3) Rinse slide with water to remove excess iodine solution.
C) Rinse the slide with Decolorizing Agent (Gram III, acetone-alcohol), until the runoff
from the smear is almost clear (no purple tint)_usually 3-5 seconds.
1) The Gram+ bacteria will retain the crystal violet stain, thus will appear purple.
2) The Gram- bacteria will not retain the crystal violet stain, thus will appear colorless.
3) Rinse slide with water to remove decolorizing agent.
The following step (v.) could be omitted and the gram-stain reaction would be known. One
would observe purple (+) and/or colorless (-) bacteria. However, colorless bacteria are very
difficult to see; therefore, we choose to add safranin.
v.
Apply the Counterstain (Gram IV, safranin): 45-60 sec.
A) The Gram+ bacteria will not take this stain and will remain purple.
Review for Mid-Term Exam – Summer 2008
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Microbiology – B. Miller
B) The Gram- bacteria can take this stain and will become pink.
C) Rinse slide with water to remove safranin stain.
After this final step, one will observe purple (+) and/or pink (-) bacteria.
*The Gram Stain is meant to be used on bacterial cultures that are 18-24 hours old. As cultures
age, particularly Gram+ cultures, some of the cells tend to take the safranin stain and appear as
Gram- and the whole smear of Gram+ organisms will be Gram variable.
d. Observation using the oil-immersion lens (100x)
A) Locate the sample on the high power (43x) lens, and focus the lens with the fine adjustment
knob.
B) Rotate the nosepiece so that condenser lens is between the high-power and the oilimmersion lens.
C) Place one small drop of oil (glycerol, also called glycerin and glycerine) on the slide in the
center of the circle of light coming from the condenser lens.
D) Rotate the nosepiece so that the oil-immersion lens is in place in the oil.
E) Use the fine-adjustment know to focus the lens on the bacteria on the slide.
3. Pp. 29-34. Hanging Drop Preparation (Wet Mount)
a. Purpose of procedure: to observe organisms in a living, unstained state.
i.
This allows the observation of motility of those organisms that are motile.
ii.
(Distinguish true motility from Brownian movement.)
motililyBrownian movement-
b. Procedure
i.
Put a drop of the suspension containing the microorganism near the center of a cover slip.
ii.
Place the cover slip on top of a depression slide with the drop of suspension hanging down
into the center of the depression.
iii.
Place the slide on the microscope and use low power (10X) lens, then the high power
(40X) lens, to observe the organisms.
c. Other considerations.
i.
What special microscope adjustments may be required to find unstained organisms?
ii.
Can stains be added to the suspension?
iii.
How can motile organisms that quickly swim out of the field of vision be slowed down?
4. Pp. 55-74. Physical Factors affecting Growth & Techniques for growing anaerobes.
a. Pp. 55-58. Temperature Requirements
i.
Purpose: to understand the different temperature ranges of bacterial enzymes.
ii.
Know these terms:
A) Minimum growth temperature
B) Maximum growth temperature
C) Optimum growth temperature
iii.
Distinguish among these types of organisms
Psychrophiles
Mesophiles
Thermophiles
Facultative thermophiles
Obligate thermophiles
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Microbiology – B. Miller
iv.
Classifiy Serratia marcescens using the above groupings. Explain how Serratia
marcescens visually illustrates optimum growth temperature.
v.
Procedure:
A) Use a wax pencil to divide the underside of 4 TSA plates into quadrants.
B) Write the names of the four organisms to the tested in the quadrants on each plate.
1) E. c. (for Escherichia coli)
2) B. s. (for Bacillus stearothermophilus)
3) P. s. (for Pseudomonas savastanoi)
4) S. m. (for Serratia marcescens)
C) Label the covers (tops) of the plates with your name and class period (8 am; 11 am)
D) Label the cover of one plate with 4°C, one with 25°C, one with 37°C, and one with 60°C.
E) Inoculate the quadrants of each plate with the organisms called for.
F) Place the plates in the incubators at the temperatures called for.
G) After 24-48 hours record the growth using this scale:
0
no growth
+
slight growth
++
moderate growth
+++
maximum growth
Organism
Temp. Class
Growth
Pigment
Gas
(See 4. a. iii.)
Escherichia coli
Bacillus stearothermophilus
Pseudomonas savastanoi
Serratia marcescens
4°
25°
37°
60°
b. Pp. 59-62. pH of extracellular environment
i.
Purpose: to observe the effects of environmental pH on the activities of bacterial enzymes.
ii.
Normal pH Ranges:
A) Bacteria tend to grow between pH 4 - 9, with the optimal range between 6.5 and 7.5.
B) Fungi, such as yeasts and molds, tend to grow between pH 4 - 6.
C) That is, average pH in the bacterial environment tends to center around pH 7 (neutral),
while average pH in the fungal environment tends to center around pH 5 (acid).
iii.
Acid-base Buffers are frequently added to culture media to prevent large departures from
specified pH.
A) Buffer Systems typically contain the salt of a weak acid (KH2PO4) and the salt of a weak
base (K2HPO4). These salts react with acids and with bases as follows:
1) K2HPO4 + HCl (acid)  KH2PO4 + KCl (thus the acid is removed)
2) KH2PO4 + KOH (base)  K2HPO4 + H2O (thus the base is removed)
B) Proteins (and amino acids, polypeptides, and peptones) have a special nature that allow
them to function as natural buffers.
H
|
1) An amino acid is a zwitterion (dipolar ion) H2N – C – COOH
|
R
2) An amino acid can accept another H at the H2N (amino group) to become H3N –, or it
can donate a H from the COOH (carboxyl group) to become –COO.
Review for Mid-Term Exam – Summer 2008
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Microbiology – B. Miller
iv.
Classification of microbes based on their optimal environmental pH. Define these terms:
Acidophile
Neutralophile
Alkalophile
c. Pp. 63-68. Atmospheric O2 Requirements
i.
Purpose: To observe the effect of free (atmospheric) oxygen on the function of bacterial
enzymes.
ii.
Classification of microbes according to oxygen requirements
A) Obligate Aerobes
1) Enzymes systems require the use of O2 as the final hydrogen/electron acceptor in the
complete oxidation of glucose.
2) They must have atmospheric O2.
B) Microaerophiles
1) Require limited amounts of atmospheric O2 for growth.
2) Excess O2 kills them.
C) Facultative anaerobes
1) Normally grow as aerobes.
2) They have the faculty of growing in O2 deprived environment; that is, they can use
molecules other than O2 as the final hydrogen/electron acceptor, or they can use an
alternate fermentative pathway.
D) Aerotolerant anaerobes
1) Use molecules other than O2 as the final hydrogen/electron acceptor.
2) These organisms possess enzymes (superoxide dismutase and catalase) to destroy the
free radicals; therefore, they are not killed by the presence of O2.
E) Obligate Anaerobes
1) Use molecules other than O2 as the final hydrogen/electron acceptor.
2) Atmospheric O2 results in the formation of poisonous oxygen radicals (see book).
3) These organisms lack the enzymes (superoxide dismutase and catalase) to destroy the
free radicals; therefore, small amounts of atmospheric oxygen are poisonous to these
organisms.
F) Interpretation of bacterial growth in sterile infusion agar.
1) See Fig. 1, page 65.
2) Be able to correctly identify an organisms O2 requirement by looking at these drawings.
d. Pp. 69-74. Techniques for cultivation of Anaerobic Microorganisms
i.
Fluid thioglycolate medium
A) HSCH2CO2H (thioglycolic acid) is readily oxided to [SCH2CO2H]2 (corresponding
disulfide of thioglycolic acid). Thus it can remove O2 from the culture medium.
B) What does a pink color in the medium indicate?
C) Why must the medium be heated, when the pink color is present?
D) Why must the thioglycolate medium be inoculated to its depth?
ii.
GasPak System
A) The GasPak System was designed to quickly and easily create an anaerobic environment.
B) Why must one ensure that the seal between the lid and container of this system is in good
order?
Review for Mid-Term Exam – Summer 2008
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Microbiology – B. Miller
C) What could happen if the gasket (sealing device) were left off the lid?
D) What chemical reaction creates the anaerobic condition in this system?
E) What is the function of the methylene blue indicator strip in the jar?
1) Blue – methylene blue is oxided … indicates the presence of oxygen
2) Colorless – methylene blue is in its reduced state … oxygen is not present.
5. Pp. 1-20, 49-54. Techniques for Isolation and Culture of Microorganisms; Use of Differential and
Selective Media
a. Pp. 1-6. Techniques for Isolation and Cultivation of Microorganisms and their Cultural
Characteristics
b. Pp. 7-14. Techniques for Isolation of pure cultures.
i.
Read pg. 7-8, 1a, 1b, 1c, 1d.
ii.
The purpose of a streak plate is to successively distribute fewer and fewer bacteria in each
section of the plate (4 of them) until separate colonies grow from each CFU (colony forming
unit).
c. Pp. 15-20. Cultural Characteristics of Microorganisms, see pg. 16, Figure 1.
i.
Types of gelatin liquefaction
Type
English description
Crateriform
Napiform
Infundibuliform
Saccate
Stratiform
ii.
Appearance of colonies on agar plates
Forms
Circular
Irregular
Rhizoid
Margins
Entire
Lobate
Undulate
Serrate
Filamentous
Growth in Broth
Uniform turbidity
Flocculent growth
Pellicle
Sediment
Growth on Agar Slants
Filiform
Echinulate
Beaded
Effuse
Arborescent
Rhizoid
Description/drawing
Description/drawing
Description/drawing
Description/drawing
d. Pp. 49-54. Differential and Selective Media
i.
Distinguish between differential and selective media.
differential media
selective media
Review for Mid-Term Exam – Summer 2008
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Microbiology – B. Miller
ii.
Some Selective Media
Médium
Organsism selected
for
Active agent and
organisms suppressed
Interpretation of
Growth
Active agents and
reactions
Interpretation of
Growth
Phenylethyl alcohol agar
Crystal violet agar
7.5% sodium chloride agar
iii.
Differential Media
Médium
Organsisms
differentiated
Mannitol salt agar
McConkey agar
Eosin Methylene Blue
agar (EMB agar)
iv.
Enriched media. One example: Blood Agar
Médium
Blood Agar
Organsisms
differentiated
Streptococci
Review for Mid-Term Exam – Summer 2008
Type of
Hemolysis
(α) alpha
(β) beta
(γ) gamma
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Interpretation of Growth
Microbiology – B. Miller