Microbiology A Laboratory Manual 11th Edition Cappuccino Solutions Manual 1

Solution Manual for Microbiology A Laboratory Manual
11th Edition Cappuccino Chad 1292176016

9781292176017
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60 Copyright © 2017 Pearson Education, Inc.

EXPERIMENTS 32 AND 33
Free-Living Protozoa
Parasitic Protozoa
These experiments are presented to give students a

brief exposure to the morphology and significance of Parasitic Protozoa
the free-living and parasitic protozoa.
Prepared Slides
Materials
Per Lab
Group Per Class
Free-Living Protozoa
E. histolytica: 1
trophozoite
Cultures
E. histolytica: cyst 1
 Stagnant pond water
 Prepared slides of amoebas, paramecia,
G. intestinalis: 1
euglenas, and stentors
trophozoite
Reagent G. intestinalis: cyst 1
 Methyl cellulose B. coli: trophozoite 1
B. coli: cyst 1
Equipment
T. gambiense 1
Per Lab
(prepared with
Group Per Class
human blood smear)
Microscope 1 P. vivax (prepared with 1

human blood smear)
Glass microscope 1
slide
Coverslip 1
Equipment
Pasteur pipette 1 Per Lab

Group Per Class
Microscope 1
Lens paper 1
Immersion oil as needed
Copyright © 2017 Pearson Education, Inc. Experiments 32 and 33 61

Procedural Point to Emphasize Answers to Review Questions
If living cultures are used for the slide preparations,
an explanation of the required use of methyl cellulose Free-Living Protozoa
should be presented.
1. The major distinguishing characteristic between

Optional Procedural Additions the classes of free-living protozoa is their mode
or Modifications of locomotion. The Sarcodina move by means of
pseudopodia, the Mastigophora via flagella, and
Stained slide preparations of the free-living protozoa the Ciliophora by means of flagella.
may be substituted for the pond water. If the intent of
these exercises is solely to introduce students to 2. a. Pseudopodia: false feet, caused by
protozoan morphology, these will facilitate cytoplasmic streaming, that are used for motility
visualization of cell structure. b. Contractile vacuole: osmoregulatory
organelle
Tips c. Eye spot: light-sensitive pigmented area
d. Micronucleus: nuclear organelle responsible
Free-Living Protozoa for sexual mode of reproduction
e. Pellicle: elastic membrane covering the cell
 Stagnant water may also be obtained from membrane
gutters, lakes, and streams. f. Oral groove: indentation leading to the
 Hay infusions may be used as a source for opening of the mouth and gullet
protozoa and should be prepared a week before 3. Individuals with AIDS possess a severely
laboratory use. suppressed immune system that allows for the
 An alternate source is to use commercially opportunistic organisms to produce infectious
prepared cultures of protozoa, but they should be processes. In the case of Pneumocystis carinii, a
fresh and received not more than 2 to 3 days life-threatening form of pneumonia develops in
before classroom use. these debilitated individuals.
Parasitic Protozoa Parasitic Protozoa
 The instructor might set up several microscopes, 1. Sporogamy represents the stage in the malarial
set the pointer on a specific structure, and name life cycle designated as the sexual cycle.
the structure on an index card placed next to the Schizogony represents the asexual phase that
microscope. occurs in the liver and blood of the human host.
2. The reduviid bug or the tsetse fly serves as the
Additional Readings invertebrate host in whom the juvenile forms
develop and give rise to the final infectious
 Lopez, C., Budge, P., Chen, J., Bilyeu, S., Mirza, trypanosomes.
A., Custodio, H., …Sullivan, K. J. (2012).
Primary amebic meningoencephalitis: A case 3. In the infected host, the pre-erythrocytic malarial
report and literature review. Pediatric Emergency stage occurs in the liver, and the erythrocytic
Care, 28(3):272–6. stage occurs in the red blood cells.
4. The sexually mature parasite, the sporozoite,
 Abdel-Hafeez, E. H., Ahmad, A. K., Ali, B. A.,
& Moslam, F. A. (2012). Opportunistic parasites resides in the salivary glands of the female
among immunosuppressed children in Minia Anopheles mosquito. This is not the case with
District, Egypt. Korean Journal of Parasitology, other protozoal parasites; only the Sporozoa
50(1):57–62. possess a sexual life cycle.
5. The migration of the amoeba into the mucosa for
nutritional purposes causes the erosion and
sloughing of the intestinal mucosa.

EXPERIMENTS 34, 35, AND 36
Cultivation and Morphology of Molds

Yeast Morphology, Cultural Characteristics,
and Reproduction
Identification of Unknown Fungi
The purpose of these mycological experiments is to Equipment

acquaint students with fungal morphology and
cultivation. This knowledge can then be applied Per Lab
toward the identification of an unknown fungal Group Per Class
organism.
Microincinerator or 1
Bunsen burner
Materials
Water bath 1
Cultivation and Morphology of Concave glass slides 4

Molds
Coverslips 4
Cultures Petroleum jelly as needed

7- to 10-day-old Sabouraud agar cultures of:
 P. chrysogenum Toothpicks as needed
 A. niger
 R. stolonifer Sterile 2-ml saline 4
 M. mucedo tubes
Sterile Pasteur pipette 1

Media
Sterile Petri dishes 4
Per Lab
Group Per Class Forceps 1
Sabouraud agar 1 Inoculating loop 1

deep tube
Inoculating needle 1
Sabouraud agar 3
plates U-shaped bent glass 4
rod
Potato dextrose agar 1
plate Thermometer 1
Dissecting microscope 1
Beaker with 95% ethyl 1

alcohol

Equipment
Yeast Morphology
Per Lab
Group Per Class
Cultures
7-day-old Sabouraud agar cultures of: Microincinerator or 1
 S. cerevisiae Bunsen burner
 C. albicans
 R. rubra Inoculating loop 1
 S. intestinalis
 S. octosporus Inoculating needle 1
Glass microscope 10
Media slides
Per Lab Coverslips 10

Group Per Class
Sterile Pasteur 5
Bromcresol purple 5 pipettes
glucose broth
w/Durham tubes Glassware marking 1
pencil
Bromcresol purple 5
maltose broth tubes Microscope 1
w/Durham tubes
Bromcresol purple 5
lactose broth tubes Identification of Unknown Fungi
w/Durham tubes
Cultures
Bromcresol purple 5
sucrose broth tubes Number-coded, 7-day-old Sabouraud broth spore
w/Durham tubes suspensions of:
 Aspergillus
Glucose–acetate 2  Mucor
agar plates  Penicillium
 Alternaria
Test tubes (13-  5
 Rhizopus
100-mm) w/ 2ml of
 Cladosporium
sterile saline
 Fusarium
 Cephalosporium
 Torula
Reagents  Candida
 Water–iodine solution
 Lactophenol–cotton-blue solution
64 Experiments 34, 35, and 36 Copyright © 2017 Pearson Education, Inc.

Media objective of this exercise is solely to acquaint
students with fungal structure.
Per Lab
Group Per Class
Tips
Sabouraud agar 1
plate
Cultivation and Morphology of
Molds
Reagent
 Lactophenol–cotton-blue solution  Petri plate or agar slant cultures are slow-
growing molds and should be prepared about 7
to 10 days prior to student use. Rhizopus cultures
Equipment grow faster than the previously mentioned
organisms and can be prepared about 3 to 5 days
Per Lab prior to class use.
Group Per Class
 Petroleum jelly can be softened (liquefied) by
heating in a hot waterbath. A Q-tip or fine-point
Bunsen burner brush may be used to coat the edges of the
coverslip on three sides. The fourth side is left
Dissecting 1 open to the atmosphere.
microscope
Hand lens 1
Yeast Morphology
Glass microscope 1  Glucose–acetate agar is one of the media used to

slide stimulate yeast sporulation. An alternate medium
that can be used is a piece of sterile carrot in a
Coverslip 1 culture tube. A yeast suspension is employed to
inoculate this medium.
Sterile cotton swabs as needed
 Selenotila intestinalis does not sporulate.
Glassware marking 1
 Saccharomyces cerevisiae produces four
pencil
ascospores in the ascus.
 Schizosaccharomyces octosporus produces eight
Procedural Points to ascospores in the ascus.
Emphasize
1. A brief review of fungal morphology, growth Additional Readings
requirements, and specialized mode of  Shah, P. D. & Deokule, J. S. (2007). Isolation of
cultivation should be presented. Aspergillus nidulans from a case of fungal
2. Filter paper is moistened with sterile water to rhinosinusitis: A case report. Indian Journal of
increase the humidity in the Petri dish and also to Pathology and Microbiology, 50(3):677–8.
prevent the agar medium from drying out. The  Shi, J. Y., Xu, Y. C., Shi, Y., Lü, H. X., Liu, Y.,
filter paper should be kept moist during the Zhao, W. S., …Guo, L. N. (2010). In vitro
incubation period. susceptibility testing of Aspergillus spp. against
voriconazole, itraconazole, posaconazole,
amphotericin B and caspofungin. Chinese
Optional Procedural Additions Medical Journal (Engl), 123(19):2706–9.
or Modifications
 Leibovitz, E. (2012). Strategies for the
Commercially prepared slides may be used instead of prevention of neonatal candidiasis. Pediatrics
the specialized microtechnique procedure if the and Neonatology, 53(2):83–9.
Copyright © 2017 Pearson Education, Inc. Experiments 34, 35, and 36 65

 Saadah, O. I., Farouq, M. F., Daajani, N. A., and in an enriched nutritional environment, they
Kamal, J. S., & Ghanem, A. T. (2012). exist as yeasts.
Gastro-intestinal basidiobolomycosis in a child;
an unusual fungal infection mimicking
fistulising Crohn’s disease. Journal of Crohn’s Yeast Morphology
and Colitis, 6(3):368–72.
1. a. Budding is an asexual reproductive process
Answers to Review Questions in which a small outgrowth pinches off from the
parent cell.
b. The ascus is the portion of the fungal cell
Cultivation and Morphology of that houses the ascospores.
Molds
c. Ascospores are the four haploid nuclei
formed as a result of meiotic division. The
1. Beneficial activities of molds include the zygote is a diploid structure formed by the
production of antibiotics, wine and beer, and conjugation of two ascospores.
food products. The detrimental effects are
2. Yeast cells are classified as fungi because they
associated with fungal pathogens that cause
are eukaryotic cells containing membrane-bound
infections of the skin, hair, nails, and lungs, as
organelles (i.e., DNA enclosed in a nuclear
well as the spoilage of food and other products.
membrane). Their morphology differs from
2. Any basic complex medium can be used to other fungi in that they tend to form ovoid bodies
cultivate fungi, provided that the pH is adjusted and are nonfilamentous.
to an acidic level. However, Sabouraud agar is
3. The industrial significance of yeast cells is their
commercially formulated with the pH adjusted to
use for the production of bread, beer, alcohol,
5.6.
ciders, cheeses, and industrial enzymes.
3. a. The moistened filter paper in the Petri dish
4. Urinary and vaginal infections caused by
is used to provide a moist, humid environment
Candida albicans are of major medical
for fungal growth.
significance.
b. The U-shaped rod in the Petri dish is used to
5. Pasteurization of fruit juices prevents the growth
elevate the slide culture above the moistened
of undesired yeasts and prevents the
paper to ensure adequate air convection.
fermentation of fruit sugars to alcohol.
4. The advantage of the culture chamber is that it
6. Prolonged antibiotic therapy represses the
allows for the direct microscopic observation of
growth of the gram-negative intestinal flora and
the colonies with the mycelial and reproductive
allows the pathogenic yeast Candida albicans to
structures intact. In addition, the colonies can
grow rapidly in the intestine. From this site, it
serve as a pure culture source for subsequent
makes its way to the urogenital system, where it
studies.
is responsible for the production of severe
5. Observation of various fungi cultivated on an vaginitis.
agar plate provides the student with the ability to
7. Wild types of yeast are naturally present on grapes
observe the colonial morphology, type of hyphae
from the field and are transferred to the grape juice
(vegetative or reproductive), pigmentation,
during the crushing process. To this juice (must),
sporangiophores, conidiophores, and other
the pure wine yeast Saccharomyces ellipsoideus is
fungal structures that assist in the identification
added to begin the fermentation process. If the
of fungi.
grapes were washed before crushing, the flora of
6. In vitro, molds exhibit their normal saprophytic wild yeast would be eliminated or greatly
forms; however, in vivo, at higher temperatures decreased, resulting in the production of a wine
that might be of poor quality.
66 Experiments 34, 35, and 36 Copyright © 2017 Pearson Education, Inc.

EXPERIMENT 37
Cultivation and Enumeration of Bacteriophages
The purpose of this experiment is twofold. First, it Equipment

emphasizes the necessity of using susceptible host
cells for viral replication. Second, it illustrates the Per Lab
procedure for bacteriophage enumeration that is Group Per Class
procedurally similar to the bacterial agar plate counts
in that both require the use of the serial dilution–agar Microincinerator or 1
plate technique. However, plaques, clear zones in the Bunsen burner
agar, rather than bacterial colonies, are counted for
viral enumeration. Waterbath 1
Thermometer 1
Materials
Sterile 1-ml pipettes 14
Cultures
24-hour nutrient broth cultures of: Mechanical pipetting 1
 E. coli B device
 T2 coliphage
Pasteur pipettes 5
Media Test tube rack 1
Per Lab Glassware marking 1

Group Per Class pencil
Tryptone agar plates 5

Procedural Points to
Tryptone soft agar 5
tubes, 2 ml per tube Emphasize
1. As this is the first time students will be using an
Tryptone broth tubes, 9 agar overlay preparation, this technique should
9 ml per tube
be explained from both the procedural and the
theoretical aspects.
2. The double-layered agar technique is a complex
procedure. The use of a variety of agar and broth
media, plus the intricacies of this serial dilution
procedure, requires that the students be cautioned
to properly organize and label all the materials
prior to the initiation of the experiment.
Copyright © 2017 Pearson Education, Inc. Experiments 34, 35, and 36 67

3. Students should be reminded that each soft agar variety of mutagens, as well as physical and
overlay dilution must be prepared, poured, and emotional stress-inducing factors.
swirled rapidly to prevent its solidification prior
3. During the replicative stage of the lytic cycle, the
to the completion of these manipulations.
host cell’s biosynthetic facilities are subverted
4. Students should be instructed to use care in for the sole purpose of synthesizing new phage
disposing of all media, glassware, and all other components. The maturation stage is
equipment in this experiment. Be sure that they characterized by the assembly of the phage
return these materials to the designated disposal components into complete phage particles.
area in the laboratory.
4. The soft agar overlay containing the phage
5. The reason for careful disposal is to prevent the particles and host cells is placed over the hard
spread of bacterial viruses to other areas, agar base to allow for the development of distinct
especially other strains of E. coli cultures. plaques in the presence of sufficient oxygen in
this upper layer. The uninfected bacterial host
cells multiply and form a cloudy layer on the
Additional Reading lower hard agar surface, thereby making the
 Tiwari, B. R., Kim, S., Rahman, M., & Kim, J. plaques more discernible.
(2011). Antibacterial efficacy of lytic 5. The number of phage particles in the original
Pseudomonas bacteriophage in normal and sample is determined by the number of plaques
neutropenic mice models. The Journal of formed, multiplied by the dilution factor. The
Microbiology, 49(6):994–9. product is expressed as the number of plaque-
forming units (PFUs) per ml of the initial sample.
Answers to Review Questions 6. 204 PFUs  109 = 2.04  1011 PFUs.
1. As a result of a lytic infection, the host cell dies 7. Irrespective of the method of viral release, the
following the replication, maturation, and release host cell will usually die. Naked viruses are
of the viruses. In lysogeny, the viral nucleic acid released by lysis of the host cell’s membrane.
molecule becomes integrated into the genome of Enveloped viruses exit the host cell by budding,
the host cell. The integrated virus, a prophage, a process that does not disrupt the host’s cell
remains as such until it is released from the host’s membrane. However, considering the host cell’s
genome to initiate the lytic cycle. facilities have been subverted for viral
2. The transformation of a lysogenic infection to replication, its own metabolic activities are
one that is lytic may be caused by inducing inhibited, and this generally leads to the death of
agents such as x-rays, ultraviolet rays, and a the cell.

EXPERIMENT 38
Isolation of Coliphages from Raw Sewage
This experimental procedure is designed to Equipment

demonstrate the presence of viruses outside of host
cells. As sewage is replete with a large variety of Per Lab
microbial forms, the viral particles are present in low Lab One Group Per Class
concentrations. Therefore, this exercise requires the
use of enrichments, namely susceptible host cells, to 250-ml Erlenmeyer 1
increase their number in order to facilitate viral flask and stopper
isolation and laboratory cultivation.
Per Lab
Materials Lab Two Group Per Class
Cultures
Sterile membrane 1
Lab One filter apparatus
 5 ml of 24-hour nutrient broth culture of E. coli B
 45-ml samples of fresh sewage collected in Sterile 125-ml 1
screw-cap bottles Erlenmeyer flask
and stopper
Lab Two
125-ml flask 1
 10 ml of 24-hour nutrient broth culture of E.
coli B
1000-ml beaker 1
Media Microincinerator or 1
Bunsen burner
Per Lab
Lab One Group Per Class Forceps 1
5-ml tube of 1 1-ml sterile 1

bacteriophage disposable pipette
nutrient broth (10 
normal) Sterile Pasteur 1
pipette
Per Lab Mechanical pipetting 1

Lab Two Group Per Class device
Tryptone agar plates 5 Glassware marking 1

pencil
3-ml tubes of 5
tryptone soft agar
Copyright © 2017 Pearson Education, Inc. Experiment 37 69

Equipment (continued) filter apparatus for the class. Then dispense about
2 to 3 ml of the filtered supernatant to each
Per Lab designated student group.
Lab Two Group Per Class
 If the previous options are not acceptable, the
instructor may obtain a commercially prepared
Centrifuge 1
phage culture along with its susceptible E. coli
host strain.
Test tube rack 1
Disposable gloves 1 pair/ Additional Reading

student
 Haramoto, E., Katayama, H., Asami, M., &
Akiba, M. (2012). Development of a novel
Procedural Points to method for simultaneous concentration of
viruses and protozoa from a single water sample.
Emphasize Journal of Virological Methods, 182(1–2):62–9.
1. The use of the enrichment culture technique is an
integral part of this experiment. As such, the Answers to Review Questions
application of this cultural procedure for the
isolation of coliphages should be presented. 1. Enrichment of sewage samples is essential to
increase the number of phage particles, which are
2. Because the membrane filter apparatus is to be
present in low concentrations in this test sample.
used for the first time, its assembly and use
should be discussed. 2. A sewage sample is enriched by the addition of a
fresh culture of susceptible host cells to
3. Students should be apprised of the fact that
increase the number of phage particles for their
sewage may contain potential pathogens.
subsequent isolation.
Therefore, the use of good aseptic techniques is
imperative during the performance of the entire 3. Sterile phage particles are obtained by the
procedure. Disposable gloves should be worn removal of gross particulates from incubated
when handling raw sewage. cultures by centrifugation and the subsequent
passage of the supernatant through a bacteria-
tight filter.
Tips
4. It is absolutely essential to exercise aseptic
 The instructor may opt not to use sewage for this techniques when handling raw sewage because of
experiment. In this case, alfalfa may serve as a the possibility of autoinfection. Sewage may
source for isolation of bacteriophage. contain a variety of enteric pathogens as well as
 The enrichment part of the experiment may be pathogenic viruses, such as the hepatitis A virus.
done as a demonstration, using the membrane

EXPERIMENT 39
Propagation of Isolated Bacteriophage Cultures
This experimental procedure is designed to Equipment

demonstrate techniques required for the propagation
and enumeration of previously cultured Per Lab
bacteriophage plaques. This technique will utilize Lab One Group Per Class
simple diffusion as a means to transfer viruses from
agar to a liquid media. Glass Pasteur 1
pipette with bulb
Materials 1.5-ml centrifuge 1

tubes
Cultures
Lab One Micropipette with tips 1
 Agar plates from Experiments 37 or 38 (1 ml)
Lab Two
 10 ml of 24-hour nutrient broth culture of E. Per Lab
coli B Lab Two Group Per Class
Media Bunsen burner

Lab One Group Per Class device
5-ml tube Tris Buffered 1 Micropipette with tips 1

Saline (TBS) (1 ml)
Glassware marking 1
Per Lab pencil
Lab Two Group Per Class
Waterbath 1
Tryptone agar plates 5
Test tube rack 1
2-ml tubes of tryptone 5
soft agar Disposable gloves 1 pair/
student
0.9-ml tubes of 9
tryptone broth

Procedural Points to to take place the higher the yield of viruses may
be.
Emphasize
1. When choosing bacteriophage plaque to perform
experiments with on day 1, choose plaques that
Additional Reading
are isolated.  Cheepudom, J., Lee, C-C., Cai, B., & Meng, M.
(2015). Isolation, characterization, and complete
2. Ensure that glass pipette goes through agar to the
genome analysis of P1312, a thermostable
bottom of the Petri dish, twist to dislodge agar.
bacteriophage that infects Thermobifida fusca.
3. Bacteriophages will be isolated from the agar Frontiers in Microbiology, 6:959. PMC. Web.
and suspended in the TBS by simple diffusion, 17 November.
but the cold temperatures that the tubes are stored
at after day 1 will slow this diffusion but is
necessary to limit further bacterial growth. Answers to Review Questions
1. Answer will be based on calculation of dilution
time the number of plaques counted.
Tips
2. No sewage samples may have multiple
 The instructor may opt not to use previously
bacteriophages strains, further methods to
plated plaques but may choose to prepare a new
identify the strains present could include viral
plate for this lab.
DNA sequencing or viral DNA isolation with
 Since this technique relies on diffusion in cold restriction digest comparisons.
temperatures, the longer the diffusion is allowed

Physical Agents of Control: Moist Heat

Physical Agents of Control:
Electromagnetic Radiations
The purpose of these experiments is twofold. First, Media

they illustrate the injurious effects of physical agents
that are commonly used to control microbial growth. Per Lab
This inhibition of microbial growth is predicated on Group Per Class
the action of these agents on vulnerable cellular
targets. The application of moist heat acts to destroy Nutrient agar plates 5
cellular enzymes. Ultraviolet, a form of
electromagnetic radiation, is especially damaging to Sabouraud agar 5
the genetic material in the cell. The second objective plates
of these exercises is to demonstrate differences in
microbial susceptibility to destruction by the 10-ml tube of nutrient 1
application of these physical agents of control. broth
Materials Equipment
Per Lab
Moist Heat Group Per Class
Cultures Microincinerator or 1
48- to 72-hour nutrient broth cultures (50 ml per 250 Bunsen burner
ml in an Erlenmeyer flask) of:
 S. aureus Waterbath (800-ml 1
beaker)
 B. cereus
72- to 96-hour Sabouraud broth cultures (50 ml per Tripod 1
250 ml in an Erlenmeyer flask) of:
 A. niger Wire gauze screen 1
 S. cerevisiae w/heat-resistant pad
Thermometer 1
Inoculating loop 1
Glassware marking 1
pencil
Sterile test tube 4

Electromagnetic Radiations Environmental Osmotic Pressure
Cultures Students should be told that Halobacterium

salinarium is the organism of choice for this
24- to 48-hour nutrient broth cultures of: experiment because it is a true halophile. Salted
 S. marcescens meats, fish, and hides, if contaminated with this
 B. cereus organism, are subject to spoilage. This organism is
Sterile saline spore suspension of: found in environments with high salt concentrations,
 A. niger such as salt lakes.
Media Electromagnetic Radiations

Per Lab
Group Per Class
Students should be made aware of the penetrating
capacity of ultraviolet radiation. In this regard,
Nutrient agar plates 7 students must be warned not to look directly into the
ultraviolet source as this radiation will produce
corneal damage. Advise students to use protective
glasses and gloves. However, they must be reminded
Equipment to remove the Petri dish covers on all plates, except
the 7-minute control plate, during each of the
Per Lab
irradiation periods.
Group Per Class
Microincinerator or 1 Optional Procedural Additions

Bunsen burner
or Modifications
Inoculating loop 1 To conserve valuable laboratory time, it is suggested
that the students be separated into three working
Ultraviolet radiation 1 groups. Each group should be assigned the task of
source inoculating one of the experiments for its joint
observation during the next laboratory session.
Glassware marking 1
pencil
Additional Readings
Disposable gloves 1 pair/
student  Rodriguez-Palacios, A. & Lejeune, J. T. (2011).
Moist-heat resistance, spore aging, and
superdormancy in Clostridium difficile. Applied
Procedural Points to and Environmental Microbiology, 77(9):3085–
91.
Emphasize
 McMeechan, A., Roberts, M., Cogan, T. A.,
Jørgensen, F., Stevenson, A., Lewis, C., …
Moist Heat Humphrey, T. J. (2007). Role of the alternative
sigma factors sigmaE and sigmaS in survival of
Salmonella enterica serovar Typhimurium
The outlined procedure is time consuming and
during starvation, refrigeration and osmotic
requires patience on the part of the students. It is
shock. Microbiology, 153(Pt 1):263–9.
imperative that the students be apprised of the
importance of maintaining the required temperature  Park, D. K., Bitton, G., & Melker, R. (2006).
during each of the prescribed heating periods. Microbial inactivation by microwave radiation in
the home environment. Journal of
Environmental Health, 69(5):17–24.
74 Experiments 40 and 41 Copyright © 2017 Pearson Education, Inc.

Answers to Review Questions higher temperatures because of the presence of
waxes and mycolic acid in the cell wall.
Moist Heat
Electromagnetic Radiations
1. Low temperatures produce a microbistatic
effect because of a decrease in the rate of 1. When gamma rays and x-rays pass through
metabolic activities. On the other hand, matter as ionizing radiations, they cause
temperatures above the maximum growth excitation and loss of electrons from molecules
temperature irreversibly denature enzymes, in their paths, thereby altering their chemical
resulting in the death of the cell. structure and activity. Also, free radical
formation occurs because of the radiation-caused
2. Tyndallization, free-flowing steam, is preferable breakdown of water and the subsequent
for the sterilization of heat-sensitive materials. formation of hydrogen peroxide, which is highly
Autoclaving, steam under pressure, is preferable toxic to cells.
when heat stability is not a problem, and in this
way, sterilization is accomplished rapidly. 2. Ultraviolet radiation cannot be used as a
sterilization agent because of its inability to
3. Cytoplasm: alteration of its colloidal state results penetrate into matter. Thus, it can only be used
from denaturation of cytoplasmic proteins. for surface disinfection. X-ray radiation, because
Cell wall: cell-wall lysis results in the formation of its shorter wavelengths and therefore greater
of an osmotically vulnerable protoplast or penetration capability, can be used for
inhibition of cell-wall synthesis. sterilization.
Nucleic acid: breakage or distortion of the DNA 3. Ultraviolet radiation causes thymine
molecule interferes with its replication and its dimerization, chemical bonding between two
role in protein synthesis. adjacent thymine molecules on one DNA strand.
This causes distortion of the DNA molecule and
Cell membrane: lysis or loss of its selective inhibits its replication, as well as the
permeability transcription, translation, and protein
4. Pasteurization is not a means of sterilization. It synthesizing functions of the cell.
utilizes lower temperatures and destroys only 4. Non-spore-forming S. marcescens is more
potential pathogens in the product without susceptible to the damaging effects of ultraviolet
altering its palatable quality. radiation than the spore-forming B. cereus. The
5. Bacillus cereus is more resistant to heat than A. latter organism is radiation resistant because of
niger because the spores of the latter are the high concentration of sulfur-containing
reproductive structures, whereas the B. cereus amino acids in the proteins of its spore coats that
spores are the result of the structural and trap the radiation, thereby protecting the DNA in
chemical transformations of the vegetative form the core of the spore.
that are intended for the survival of the 5. Shielding of the hands from ultraviolet light is not
organisms. required because the uppermost layer of the skin
6. Aerobic and anaerobic spore formers are more is composed of fully keratinized dead cells that
heat resistant than the tubercle bacillus because of prevent the penetration of this radiation into the
the presence of calcium and dipicolinic acid in the underlying living tissues. On the other hand, the
spore cortex. The tubercle bacillus may tolerate cornea is composed of viable cells that can be
destroyed by exposure to ultraviolet radiation.

EXPERIMENT 42
Chemical Agents of Control:

Chemotherapeutic Agents
Both of the methodologies presented in this

Per Lab
experiment are of clinical significance. The Kirby- PART B Group Per Class
Bauer procedure is routinely used to rapidly
determine the antibiotic of choice for the treatment of Mueller-Hinton agar 4
a microbial infection. The second procedure is plates
intended to illustrate the efficacy of using drug
combinations to enhance the antimicrobial activity of
antibiotics.
Antimicrobial Sensitivity Discs
Materials Per Lab

PART A Group Per Class
Cultures
Penicillin G, 10 μg 7
PART A
0.85% saline suspensions adjusted to an A equal to
Streptomycin, 10 μg 7
0.1 at 600 nm:
 E. coli Tetracycline, 30 μg 7
 S. aureus
 P. aeruginosa Chloramphenicol, 7
 P. vulgaris 30 μg
 M. smegmatis
 B. cereus Gentamicin, 10 μg 7
 E. faecalis
PART B Vancomycin, 30 μg 7
0.85% saline suspensions adjusted to an A equal to
0.1 at 600 nm: Sulfanilamide, 300 μg 7
 E. coli
 S. aureus
Per Lab
PART B Group Per Class
Media
Tetracycline, 30 μg 2
Per Lab
PART A Group Per Class Trimethoprim, 5 μg 4
Mueller-Hinton agar 7 Sulfisoxazole, 150 μg 2

plates

Equipment medium must be standardized to a pH of 7.2 to
7.4, poured to a depth of 5.0 mm, and dried in an
Per Lab incubator for 15 minutes prior to its use.
 Petri dishes measuring 150 mm are
recommended to accommodate a greater number
Sensi-DiscTM 1
of antibiotic-impregnated discs. Use of Sensi-
dispenser or forceps
Disc dispensers is preferable for the equidistant
Millimeter ruler 1
placement of the antibiotic discs on the agar
surface.
Bunsen burner PART A: Kirby-Bauer Procedure
Cotton swabs as needed  Because Sensi-Disc dispensers are expensive
and may not be available, antibiotic-impregnated
Glassware marking 1 discs may be applied to the surface of the seeded
pencil agar plates with a sterile forceps.
 Time and materials could be conserved by
having groups of four or more working on each
Per Lab
PART B Group Per Class set of bacterial species and antibiotics.
 Antibiotic Sensi-Discs™ should be stored in the
Microincinerator or 1 refrigerator.
Bunsen burner
Forceps 1 PART B: Synergistic Effects of Drug

Combinations
Sterile cotton swabs as needed
 It should be emphasized that students should
Millimeter ruler 1 stringently adhere to the required distance
between the points for the placement of the
Glassware marking 1 antibiotic-impregnated discs.
pencil
Additional Readings
Procedural Points to  Medell, M., Medell, M., Martínez, A., & Valdés,
R. (2012). Characterization and sensitivity to
Emphasize antibiotics of bacteria isolated from the lower
PART A: Kirby-Bauer Procedure respiratory tract of ventilated patients
hospitalized in intensive care units. Brazilian
1. To ensure a confluent lawn of microbial growth, Journal of Infectious Diseases, 16(1):45–51.
students should be reminded to inoculate the
entire plate in both a horizontal and a vertical  Rand, K. H., & Houck, H. J. (2004). Synergy of
direction. daptomycin with oxacillin and other beta-lactams
against methicillin-resistant. Staphylococcus
2. If forceps are used for disc placement, students aureus. Antimicrobial Agents in Chemotherapy,
must be cautioned to flame the forceps between 48(8):2871–5.
the applications of the discs, and care must be
taken to place them at a distance from each other.
3. Students must be cautioned to gently press the
Answer to Review Question
discs onto, not into, the agar surface. 1. These broad-spectrum antibiotics exert their
antimicrobial effect on the 70s functional
ribosome of prokaryotic cells, thereby interfering
Tips with the process of protein synthesis. Eukaryotic
 Mueller-Hinton agar must be used for the proper cells possess an 80s functional ribosome that is not
interpretation of the zones of inhibition. The a cellular target for these antibiotics.
Copyright © 2017 Pearson Education, Inc. 77

EXPERIMENT 43
Determination of Penicillin Activity in the

Presence and Absence of Penicillinase

illustrates the use of the broth culture dilution
system to determine the minimal inhibitory Per Lab
concentration (MIC) of an antibiotic. Second, it Group Per Class
demonstrates the enzymatic basis for antibiotic
resistance in microorganisms. Two methods are Microincinerator or 1
presented, the first utilizing a spectrophotometer and Bunsen burner
the second utilizes a plate reader.
Sterile 13-  100-mm 20
test tubes
Materials
Test tube racks 2
Cultures
1:1000 brain heart infusion (BHI) broth dilutions of Sterile 96-well plate 1
24-hour BHI broth cultures of:
 S. aureus ATCC® 27661™ (penicillin-sensitive Sterile 2-ml pipettes 5
strain)
 S. aureus ATCC 27659 (penicillinase-producing Sterile 10-ml pipette 1
strain)
Mechanical pipetting 1
device
Media
Spectrophotometer 1
Per Lab
Group Per Class Colorimetric plate NA 1
reader
40 ml brain heart 1
infusion in 100-ml Glassware marking 1
Erlenmeyer flask pencil
10 ml sterile aqueous 3 Disinfectant solution 1

crystalline penicillin G in a 500-ml beaker
78 Experiment 42 Copyright © 2017 Pearson Education, Inc.

Procedural Points to penicillinase detection in Staphylococcus
aureus. Clinical Microbiology and Infection,
Emphasize 14(6):614–6.
1. Although students should be familiar with serial
dilution procedures, a brief review of the twofold
dilution used in this experiment would be
Answers to Review Questions
helpful. 1. Yes. Genetically, some bacterial strains were
capable of beta-lactamase synthesis. However,
2. Penicillin solutions should be refrigerated when
this enzyme was not produced until after the
not in use. Frozen aqueous samples retain their
introduction of penicillin. With the early
potency for at least 30 days.
indiscriminate use of this antibiotic, selection
3. Many microorganisms other than S. aureus are favored the survival of the beta-lactamase-
genetically programmed to produce penicillinase producing strains, which are now predominant in
(beta-lactamase). As such, students should be nature.
instructed to use good aseptic technique to
2. At the MIC end point, some viable cells may still
prevent contamination of media.
be present. The minimal bactericidal
4. Well A1 should be used as negative controls with concentration (MBC) may be used to determine
highest concentration of antibiotic and bacteria, the bactericidal level of an antibiotic. To perform
while well A12 should be positive control wells this procedure, a 0.5-ml test sample of each MIC
with bacteria and media without antibiotic. culture that showed no growth is placed into 12
ml of molten brain heart infusion agar. The
cultures are then mixed, pour-plate preparations
Additional Reading are made, and colony counts are performed
 Kaase, M., Lenga, S., Friedrich, S., Szabados, F., following incubation. The MBC is the lowest
Sakinc, T., Kleine, B., & Gatermann, S. G. concentration of the antibiotic that resulted in
(2008). Comparison of phenotypic methods for no growth on the agar plate.

EXPERIMENT 44
Chemical Agents of Control:

Disinfectants and Antiseptics
This experiment presents two screening procedures to Disinfectants/Antiseptics

determine the efficacy of antimicrobial agents for use
in disinfection and asepsis. The agar plate–sensitivity Per Lab Per
method is procedurally similar to the filter paper Group Class
disc–agar diffusion method for the determination of
antibiotic sensitivity. However, this is a qualitative 10 ml of tincture of 1
procedure and is not as standardized as the Kirby- iodine in a 25-ml beaker
Bauer method. The second method is a modified
version of the industry standard use dilution test. This 10 ml of 3% hydrogen 1
modified technique examines the ability of a peroxide in a 25-ml
chemical to disinfect a contaminated surface. beaker
10 ml of 70% isopropyl 1
Materials alcohol in a 25-ml
beaker
Cultures
24- to 48-hour Trypticase soy broth cultures of: 10 ml of 5% chlorine 1
bleach in a 25-ml
 E. coli
beaker
 S. aureus
 B. cereus
 M. smegmatis
Equipment
7-day-old Trypticase soy broth culture of:
 B. cereus Per Lab Per
Group Class
Media Sterile Sensi-Discs in 20

4 different colors
Per Lab
Group Per Class Sterile glass slides or 25
coverslips
Trypticase soy agar 5
plates Forceps 1
50-ml tubes 25 Sterile cotton swabs as needed

containing 20 ml
of tryptic soy broth
Glassware marking 1
each
pencil
Bunsen burner

Procedural Points to the agents may be tested over a range of
concentrations to determine the correlation of
Emphasize concentration to the degree of effectiveness of the
Students should be reminded of the following: agent.
1. Inoculate the agar plates in the manner

described in Experiment 41 to obtain a confluent Additional Reading
lawn of microbial growth.
 Hosseini, H., Ashraf, M. J., Saleh, M.,
2. Following their saturation, the impregnated discs Nowroozzadeh, M. H., Nowroozizadeh, B.,
must be drained prior to their placement on the Abtahi, M. B., …Nowroozizadeh, S. (2012).
agar surface to ensure the formation of regular Effect of povidone-iodine concentration and
margins on the zones of inhibition. exposure time on bacteria isolated from
endophthalmitis cases. Journal of Cataract &
3. Students should record the color of the Sensi-
Refractive Surgery, 38(1):92–6.
Disc for each disinfectant in their notebooks.
4. Students should allow the glass slides or
coverslips to fully air dry submerging in Answers to Review Questions
solutions, the touch slide/coverslip to paper 1. Factors such as the toxicity of the chemical and
towel to wick of excess antiseptic solution. environmental conditions must be considered
before arbitrarily changing the exposure time.
Optional Procedural Additions 2. The term germicidal implies that the
or Modifications antimicrobial agent is microbicidal. However, it
does not specifically indicate the type or types of
The antimicrobial test agents may be varied microbes the agent will effectively kill.
according to students’ personal interests. In addition,

EXPERIMENT 45
Microbiological Analysis of Food Products:

Bacterial Count
The purpose of this experiment is to illustrate a Equipment

methodology that is used to determine the
microbiological quality of food products. Quality in Per Lab
this case does not imply sterility. What is important Group Per Class
in the quality testing of food is the number and types
of its endogenous microbial population. The Bunsen burner 1
procedure for the microbiological analysis of food
products is concerned with the determination of the Waterbath 1
number of bacteria present in commercial food
products, as well as the detection of E. coli, which is Quebec or electronic 1
one of the major indicators of possible contamination colony counter
by enteric pathogens.
Sterile glassine as needed
weighing paper
Materials
Blender w/three 1
Cultures sterile jars
 20 g of fresh vegetables
 20 g of ground beef Sterile Petri dishes 9
 20 g of dried fruit
1-ml pipettes 6
Media Mechanical pipetting 1

device
Per Lab
Group Per Class Inoculating loop 1
15-ml brain heart 9 Glassware marking 1

infusion agar deep pencil
tubes
Balance 1
Eosin-methylene 3
blue (EMB) agar
plates Procedural Point to Emphasize
99-ml sterile water 3 Both procedures are easy to perform. The only
blanks required specialized techniques are the serial dilution
and the pour-plate procedures, both of which students
180-ml sterile water 3 have encountered in previous experiments.
blanks

Optional Procedural Additions refreezing of certain products, and from infected
food sources, such as poultry, shellfish, and milk.
or Modifications
2. It is not advisable to thaw and refreeze foods, as
1. It is suggested that the test samples be endogenous microorganisms may multiply
homogenized prior to the start of the laboratory rapidly at room temperature. If gram-negative
session as a time-saving step. organisms are present, they may lyse upon
2. One of the test samples may be seeded with an refreezing, thereby liberating the endotoxic
enteric organism to simulate a contaminated food lipopolysaccharides in their cell walls that are
product. heat stable and not destroyed during cooking of
the food.
Additional Reading 3. Any of the foods consumed by the students may

be the source of the staphylococcal food
 Kase, J. A., Borenstein, S., Blodgett, R. J., & poisoning. Ham and sour pickles, because of the
Feng, P. C. (2012). Microbial quality of bagged higher salt concentrations, favor staphylococcal
baby spinach and romaine lettuce: Effects of top growth. Potato salad and cream puffs, because of
versus bottom sampling. Journal of Food their high carbohydrate content, can serve as
Protection, 75(1):132–6. vehicles for contamination by a variety of
microorganisms, including the staphylococci.
The elevated summer temperatures foster rapid
Answers to Review Questions multiplication of the staphylococci with the
1. The contamination of foods with enteric elaboration of the enterotoxin into the foodstuffs.
organisms may occur via infected food handlers, It is this exotoxin that is responsible for the
during the commercial processing, thawing, and intestinal symptomatology.

EXPERIMENT 46
Microbial Fermentation
The purpose of this experiment is to familiarize Equipment

students with the microbial fermentations used in the
production of wine. Methods will examine the Per Lab
processes involved in both alcohol and lactic acid Group Per Class
fermentation by microbes.
1-L Erlenmeyer flask 1
Materials One-holed rubber 1

stopper w/2-in glass
Culture
tube w/cotton
 50 ml of white grape juice broth culture of
S. cerevisiae var. ellipsoideus incubated for 400 ml stoppered 4
48 hours at 25°C graduated
Erlenmeyer flasks
 50 ml of Lactobacillus delbrueckii subsp. with stopper
bulgaricus and Streptococcus thermophiles that
are each 24 hours old. Pan balance 1
Spatula 1
Media
Glassine paper as needed
Per Lab
Group Per Class
10-ml graduated 1
cylinder
500 ml pasteurized 1
white grape juice
Burette or pipette for 1
titration
400 ml pastuerized 1
heavy cream
Ebulliometer 1
(optional)
Reagents Hot plate 1

 1% phenolphthalein solution
pH paper 1
 0.1 N NaOH
 Sucrose (table sugar)

Procedural Points to Additional Reading
Emphasize  Schuller, D., Cardoso, F., Sousa, S., Gomes, P.,
1. Fruit juices other than grape juice (e.g., apple Gomes, A. C., Santos, M. A., …Casal, M. (2012).
juice, apricot juice, pear juice, or juice from Genetic diversity and population structure of
berries) may be substituted. Likewise, Saccharomyces cerevisiae strains isolated from
dandelions or rhubarb may also be used. The different grape varieties and winemaking regions.
instructor should emphasize that wine PLoS One, 7(2):e32507.
production from any type of fruit juice requires
the correct yeast culture, sugar, and fermentation Answers to Review Questions
under anaerobic conditions.
1. Sulfite is added to the must, the first juice, to
2. The instructor should remind students that retard the growth of acetic acid–producing
commercially bought yogurt produced by lactic bacteria, molds, and wild yeasts—organisms that
acid fermentation will also contain living are endogenous to vineyard grapes.
cultures of many bacterial species.
2. During the aging process, the wine is clarified of
3. The instructor might wish to review the turbidity, thereby producing volatile esters that
glycolytic pathway, showing the enzymatic are responsible for the characteristic flavors of
production of ethanol and lactic acid. different wines.
3. The end products of this fermentation are
Optional Procedural Additions carbon dioxide and ethyl alcohol.
or Modifications 4. Red wines are produced by crushing and
This experiment can be performed as a class fermenting the red grapes along with their skins.
demonstration. The results can then be observed by White wine is produced from the juice of white
the entire class over the required 3-week period grapes. The latter are aged for a shorter period of
without detracting from the performance of other time than the red wines.
laboratory exercises. 5. The lowered pH due to fermentation will
denature milk proteins and aid in coagulation of
the milk solids to thicken the mixture.

Standard Qualitative Analysis of Water

Quantitative Analysis of Water:
Membrane Filter Method
The purpose of both experimental procedures is to Media

determine the potability of water sources. The first
method is a qualitative method, designed to detect the Per Lab
presence of coliform bacteria, indicators of fecal Lab One Group Per Class
contamination. The second technique is used to
quantitate the microorganisms that are present in a Double-strength 15
test sample. lactose fermentation
The membrane filter technique has been broth tubes
recognized and approved by the U.S. Public Health
Service for the detection of E. coli, the indicator of Single-strength 30
fecal pollution. It is a much more sensitive method of lactose fermentation
water analysis than the standard agar plate method, broth tubes
and larger volumes of water can be tested.
Per Lab
Materials Lab Two Group Per Class
Standard Qualitative Analysis of Eosin-methylene 3

Water blue agar plates or
Endo agar plates
Cultures
Lab One Per Lab
Water samples from: Lab Three Group Per Class
 Sewage
 Pond water Nutrient agar slants 3
 Tap water
Lactose fermentation 3
Lab Two broth tubes
 24-hour-old positive cultures from each of the
three series of the presumptive test
Lab Three Reagents
 24-hour coliform-positive EMB or Endo agar Lab Three
cultures from each of the three series of the
 Crystal violet
confirmed test
 Gram’s iodine
 95% ethyl alcohol
 Safranin

Equipment
Per Lab
Lab One Group Per Class
Cultures
Microincinerator or 1  Water samples collected upstream and
Bunsen burner downstream from the outlet of a sewage
treatment plant
Test tubes 45
Test tube rack 1 Media
Sterile 10-ml pipettes 3 Per Lab

Group Per Class
20-ml tube of 1
Sterile 0.1-ml 3 m-Endo broth
pipettes
20-ml tube of m-FC 1
Mechanical pipetting 1 broth
device
20-ml tube of KF 1
Glassware marking 1 broth
pencil
90-ml sterile water 4
blanks
Per Lab
Lab Two Group Per Class 300-ml flask of sterile 1
water
Bunsen burner
Equipment
Inoculating loop 1
Per Lab
Glassware marking 1 Group Per Class
pencil
Sterile membrane 1
filter apparatus
Per Lab
Lab Three Group Per Class 1-L suction flask 1
Microincinerator or 1 Sterile membrane 15

Bunsen burner filters and absorbent
pads
Staining tray 1
Sterile 50-mm Petri 15
Inoculating loop 1 dishes
Bibulous paper as needed 10-ml pipettes 12
Microscope 1 Mechanical pipetting 1

device
Glassware marking 1
pencil Small beaker of 95% 1
ethyl alcohol
Lens paper 1

Equipment (continued)  Coliforms produce a green metallic sheen on
EMB agar.
Per Lab
Group Per Class
Membrane forceps 1  The volumes of undiluted water samples to be
analyzed accurately have been determined and
Watertight plastic as needed may be found in the Standard Methods for the
bags Examination of Water and Wastewater. Some
examples are:
Dissecting 1
microscope
Water Source Volume in Milliliters
Glassware marking 1
pencil Raw sewage 0.0001 to 0.1
Waterproof tape as needed Lakes 10 to 100
44.5°C waterbath 1 Rivers 0.001 to 1.0
Disposable gloves 1 pair/ Beaches to 10

student
Drinking (tap) water 100
Procedural Points to Swimming pools 100
Emphasize Well water 10 to 100
Standard Qualitative Analysis of  If the collected water samples are not to be tested
Water immediately, they should be stored in the
refrigerator at 4°C. This reduces the possibility
of additional microbial growth.
1. Students should not confuse a single bubble of
air in the Durham tube as evidence of gas  Water samples containing large amounts of algae
formation. or visible debris should not be tested.
2. Students should use gloves when dealing with

water samples. Additional Readings
 Gronewold, A. D. & Wolpert, R. L. (2008).
Membrane Filter Method Modeling the relationship between most
probable number (MPN) and colony-forming
unit (CFU) estimates of fecal coliform
Considering this is the first time students will be concentration. Water Research, 42(13):3327–34.
using membrane filters, a demonstration and a
detailed explanation of their use, assembly, and care  Kozuskanich, J., Novakowski, K. S., &
should be given. Anderson, B. C. (2011). Fecal indicator bacteria
variability in samples pumped from monitoring
wells. Ground Water, 49(1):43–52.
Tips
Qualitative Analysis of Water
Standard Qualitative Analysis of
 If students are to collect their own water samples Water
from lakes, ponds, streams, or rivers, they should
be aware that the majority of living organisms
reside in about the first 12 inches of water.

1. E. coli is the predominant member of the resident anaerobes. In sewage, the anaerobic flora,
flora of the intestines. As such, its presence in activated sludge, acts to enzymatically degrade
water is indicative of fecal contamination and the protein matter, whereas the aerobic flora acts to
possible presence of enteric pathogens. degrade organic molecules in the water sprayed
on trickling filter beds.
2. This procedure is qualitative; it is designed to
detect the presence of specific
microorganisms—coliforms—rather than their Membrane Filter Method
number.
3. It is important to analyze water supplies serving 1. The advantages of this method are that the
industrial communities for a variety of reasons. results are available in a short period of time,
Water is used in the manufacture of numerous large volumes of a sample can be processed, and
industrial products that may become the results are readily reproducible.
contaminated by the use of polluted water. The
deposition of both medical and chemical wastes 2. The major disadvantage is the presence of large
may serve as the source of microbial and toxic numbers of gross particles, which clog the filter
pollution, destroying the potability of the water. and impede the passage of the specimen being
tested.
4. Most of the microorganisms found in natural
bodies of water represent its natural flora that 3. The FC, fecal coliforms, to FS, fecal
serves as part of the food chain for aquatic life- streptococci, ratio is used to indicate the source
forms. Microbial contaminants, deposited into of fecal pollution.
the water by lower animal species, may also be 4. The membrane filter technique may be used for
present. The microorganisms present in sewage the analysis of air (e.g., to determine pollen counts
systems are primarily enteric aerobes and and the particulate composition of smog).

EXPERIMENT 49
Microbial Populations in Soil: Enumeration

acquaints students with the diverse resident microbial
flora of the soil. Second, it quantitates the number of Per Lab
bacteria, fungi, and actinomycetes present in a soil Group Per Class
sample.
Bunsen burner
Materials
Petri dishes 12
Soil
 1-g sample of freshly pulverized, rich garden soil Quebec colony 1
in a flask containing 99 ml of sterile water, counter*
labeled 1:100 (10–2)
Mechanical hand 1
counter*
Media
Per Lab
Group Per Class Mechanical pipetting 1
device
Glycerol yeast agar 4
deep tubes L-shaped bent glass 1
rod (optional)
Sabouraud agar 4
deep tubes Turntable (optional) 1
Nutrient agar deep 4 95% ethyl alcohol in 1

tubes 500-ml beaker
99-ml flasks of sterile 2 Glassware marking 1

water pencil
*These may be replaced with an electronic colony

counter and electronic probe, if available.

Procedural Points to Dispersal of Salmonella Typhimurium by rain
splash onto tomato plants. Journal of Food
Emphasize Protection, 75(3):472–9.
1. The diversity of the microbial soil flora and the
different media used for their isolation should be
discussed.
1. No. Soil populations will vary with changes in
2. Students should be reminded that the standard
temperature, pH, soil oxygenation, and
agar plate count will only account for aerobic
availability of moisture and nutrients. These
microorganisms and not include members of the
factors will vary with the change of seasons.
anaerobic soil population.
2. Different media are needed to support the
3. For comparison purposes, the instructor may
microbial growth for the isolation of the three
wish to use a second soil sample of poorer
different types of organisms because of
quality.
differences in their nutritional and environmental
requirements.
Optional Procedural Additions 3. No. Anaerobes would not grow in a nutrient agar
or Modifications plate culture that is incubated under aerobic
conditions. The diffusion of oxygen through this
It is suggested that the class be divided into three
shallow layer of medium would either kill or
groups, each group being responsible for the isolation
inhibit the growth of these organisms.
of one of the types of soil microorganisms. The
observation of all the results can then be shared by the 4. Most microorganisms are found in the upper soil
entire class. layers because of the abundance of inorganic and
organic nutrients, moisture, and oxygen in these
layers.
Tip
5. A soil devoid of its microbial flora could not
 Soil samples must be thoroughly mixed to support plant life. These microorganisms are
provide a uniform distribution of organisms in essential for the production of fertile soil by their
the soil samples before giving them to the ability to degrade macromolecular materials into
students. the low-molecular-weight nutrients required
by plants. In turn, animal life cannot be sustained
Additional Reading in the absence of plant life.
 Cevallos-Cevallos, J. M., Danyluk, M. D., Gu,

G., Vallad, G. E., & van Bruggen, A. H. (2012).

EXPERIMENT 50
Isolation of Antibiotic-Producing Microorganisms

and Determination of Antimicrobial Spectrum
of Isolates
The purpose of this experiment is twofold. First, it

Per Lab
shows how to isolate soil organisms capable of PART B Group Per Class
excreting antibiotic substances. Second, it
demonstrates how to determine the spectrum of Trypticase soy agar 2
antimicrobial activity of the isolated antibiotics. plates
Materials
Equipment
Cultures
Per Lab
PART B
24-hour Trypticase soy broth cultures of:
 E. coli
500-ml beaker 1
 S. aureus
 M. smegmatis Test tubes 6
 P. aeruginosa
Test tube rack 1
Soil Suspensions
Sterile Petri dishes 6
PART A
 1:500 dilution of soil sample suspension (0.1 g Inoculating needle 1
of soil per 50 ml of tap water) to serve as an
unknown Hot plate 1
 1:500 dilution of soil sample seeded with
S. griseus (0.1 g of soil per 50 ml of tap water) to Thermometer 1
serve as a positive control
1-ml pipettes 3
Media 5-ml pipettes 4

PART A Group Per Class device
15-ml Trypticase soy 6 Magnifying hand lens 1

agar deep tubes

slants

(Penicillium chrysogenum is a producer of
Per Lab
PART B Group Per Class
penicillin).
Microincinerator or 1 Additional Reading

Bunsen burner
 Ding, R., Wu, X. C., Qian, C. D., Teng, Y., Li, O.,
Inoculating loop 1 Zhan, Z. J., …Zhao, Y. H. (2011). Isolation and
identification of lipopeptide antibiotics from
Glassware marking 1 Paenibacillus elgii B69 with inhibitory activity
pencil against methicillin-resistant Staphylococcus
aureus. Journal of Microbiology, 49(6):942–9.
Procedural Points to Answers to Review Questions

Emphasize
1. Antibiotics are modified in the laboratory
1. Although students are familiar with the required principally to increase their potency, to reduce
procedures for the performance of this exercise, their toxic side effects, and to circumvent their
namely the serial-dilution and pour-plate resistance to the antimicrobial agent.
procedures, the rationale for the use of the
crowded-plate technique should be discussed. 2. Antibiotics can be obtained from a variety of
microbial sources other than true bacteria, such
2. The inoculation procedure for the determination as filamentous Streptomyces and Actinomyces,
of the spectrum of antimicrobial activity should and fungi.
be explained and demonstrated to students.
3. Although a limited number of test organisms
3. Students should understand that the most prolific were used, they represent a broad spectrum of
antibiotic producers are found among the bacterial types: gram-negative, gram-positive,
Actinomyces (Streptomyces griseus produces and acid-fast organisms. However, only
streptomycin), aerobic spore formers (Bacillus antibacterial activity could be determined with
subtilis produces bacitracin, while Bacillus the use of these organisms.
polymyxa produces polymyxin), and molds

EXPERIMENT 51
Isolation of Pseudomonas Species by Means

of the Enrichment Culture Technique
The purpose of this experiment is to acquaint students Equipment

with the enrichment culture procedure used for the
isolation of a specific organism that is present in low Per Lab
numbers in a given environment. This technique has Group Per Class
numerous applications in industrial microbiology for
the isolation of specific microorganisms from soil, air, Sterile 10-ml pipette 1
and water, as well as in clinical areas for the isolation
of pathogens from biological samples. Sterile 5-ml pipette 1
Sterile 1-ml pipette 1

Materials
Culture
device
 Rich garden soil or compost sample
Microspatula 1
Media Microincinerator or 1
Bunsen burner
Per Lab
Group Per Class Staining tray 1
Erlenmeyer flasks 2 Lens paper as needed

w/20 ml of basal
salts broth w/2 ml of Bibulous paper as needed
2.5% mandelic acid
Inoculating loop 1
Agar plates of same 2
composition as
Glassware marking 1
above
pencil
Trypticase nitrate 1
Glass microscope 5
broth
slides
Litmus milk 1

slant

Reagents or Modifications
 Crystal violet 1. For allied health students, a stool sample or a
 Gram’s iodine simulated stool specimen seeded with
 95% ethanol Salmonella organisms may be substituted to
 Safranin demonstrate this technique with the use of
 Solution A (sulfanilic acid) selenite enrichment broth medium.
 Solution B (alpha-naphthylamine)
 Zinc powder 2. The instructor may opt to have individual
students gather soil samples from different
environments to be used in this experiment rather
Procedural Points to than the rich garden soil or compost.
Emphasize
1. The difference between enrichment broths, as Additional Reading
used in this exercise, and enriched media,  Lin, L. H., Tsai, C. Y., Hung, M. H., Fang,
utilized for the cultivation of fastidious Y. T., & Ling, Q. D. (2011). Rectal swab
organisms, should be discussed. sampling followed by an enrichment culture-
2. An explanation of the sequential culture based real-time PCR assay to detect Salmonella
transfers, required in the performance of this enterocolitis in children. Clinical Microbiology
experiment, should be presented. and Infection, 17(9):1421–5.
3. Students should be cautioned to handle solution

B (alpha-naphthylamine) carefully. Answers to Review Questions
4. The rationale for use of enrichment culture 1. The Salmonella organisms in the stool specimen
procedures should be explained. Indicate that may be present in low numbers. The enrichment
this type of culture is used to isolate a particular culture technique will selectively enhance the
organism from a large, complex population growth of this suspected pathogen, allowing for
found in diverse populations, in this case, soil. its isolation and the subsequent confirmation of
its presence. The use of a selective medium
5. Some relevant examples should be cited, such as
would not necessarily result in the detection of
isolation of an organism that can degrade
these organisms because of the presence of a
petroleum. In this case, the petroleum would
much larger number of other gram-negative
serve as the sole source of carbon. Isolation of
enteric competitors in the stool sample.
organisms that can break down toxic materials or
herbicides may also be discussed. 2. A basal salts medium containing the gelatinous
ascites material as the sole carbon source may be
used to isolate organisms capable of
Optional Procedural Additions enzymatically degrading this viscous substance.
This procedure represents a clinical application
of the enrichment culture technique.

EXPERIMENT 52
Enzyme Induction
The purpose of this experiment is to introduce Equipment

students to the difference between constitutive and
inducible (adaptive) enzymes. The experimental Per Lab
procedure is designed to illustrate the substrate- Group Per Class
dependent regulatory mechanism for the synthesis of
inducible enzymes. 1-ml sterile pipettes 3
5-ml sterile pipettes 2

Materials
Sterile 13-  100-mm 6
Cultures
test tubes
25-ml inorganic synthetic broth suspensions of
12-hour nutrient agar cultures: Test tube rack 1
 Lactose-positive strain of E. coli (ATCC
e23725) adjusted to an A of 0.1 at 600 nm Sterile 25-ml 6
 Lactose-negative strain of E. coli (ATCC Erlenmeyer flask
e23735) adjusted to an A of 0.1 at 600 nm
Spectrophotometer 1
Media Shaking waterbath 1

incubator
Per Lab
Group Per Class Glassware marking 1
pencil
Dropper bottle of 1
sterile 10% glucose Mechanical pipetting 1
device
Dropper bottle of 1
sterile 10% lactose
Procedural Points to
Dropper bottle of 1
sterile water Emphasize
1. An explanation of the purposes for the use of the
auxotrophic and prototrophic E. coli strains, the
Reagents three different types of media, and the substrate
analog ONPG should be presented.
 Toluene
 Orthonitrophenyl-ß-D-galactoside (ONPG) 2. It may be helpful to briefly review feedback
inhibition, enzyme repression, and enzyme
inducibility.

3. It is important that students understand that the b. Conjugation: Some gram-negative bacteria
enzymes involved in the breakdown of glucose have been shown to have a fertility (F) factor.
and energy are inducible enzymes. Those having this factor are designated as F+;
some F+ cells are further designated as high-
frequency recombinants (Hfrs). Both types are
Additional Reading considered male. Those cells lacking the F factor
 Marbach, A. & Bettenbrock, K. (2012). lac are designated as F– and are called female. The
operon induction in Escherichia coli: Systematic F+ cells are able to transmit their genetic material
comparison of IPTG and TMG induction and (DNA) to the F– via a protoplasmic tube called
influence of the transacetylase LacA. Journal of the conjugative pilus.
Biotechnology, 157(1):82–8. Conjugation between F+ and Hfr cells and F–
cells can be used to map genes and transfer
Answers to Review Questions resistance factors from F+ to the F– cells.
1. Constitutive enzymes are always present in cells, c. Transformation: This occurs when naked
regardless of their need. Inducible enzymes are DNA in the form of a plasmid (short segments of
synthesized only when they are required, in the DNA released by cell lysis from a host cell) is
presence of their specific substrates. taken up by a competent cell. Genetic
recombination occurs by the incorporation of the
2. An operon is a cluster of genes that functions as plasmid into the recipient cell’s genome. An
a unit. Transcription occurs when the inducer example would be the transformation of an
substrate is present. antibiotic-sensitive cell to one that is antibiotic
3. The use of the ONPG in this experiment is to resistant.
serve as a colorless analog of lactose, capable of 5. Penicillin is a beta-lactam antibiotic.
being hydrolyzed to galactose and a yellow Staphylococcus aureus contains a gene for the
nitrophenolate ion in the presence of beta- production of beta-lactamase (penicillinase), an
galactosidase. Therefore, the appearance of a enzyme capable of rupturing the beta-lactam
yellow color in the medium is indicative of the ring and rendering penicillin ineffective. This
synthesis of this enzyme. inducible enzyme was initially under genetic
4. a. Transduction: This process involves viral repression and not produced in the presence of
introduction of genetic material into the cell, penicillin. The apparent early indiscriminate use
resulting in genetic recombination of viral DNA of this antibiotic resulted in a mutation that
with recipient DNA. Transducing derepressed the gene, resulting in the conversion
bacteriophages have been used to map regions of this inducible enzyme into one that is
within the gene. constitutive and always present in the cell to
inactivate penicillin.

EXPERIMENT 53
Bacterial Conjugation
The purpose of this experiment is to demonstrate a Equipment

means of genetic recombination in asexually
reproducing cells. The method under investigation in Per Lab
this exercise is conjugation, which is a unidirectional Group Per Class
transfer of DNA between sexually competent donor
and recipient cells. Microincinerator or 1
Bunsen burner
Materials Beaker with 95% 1

ethyl alcohol
Cultures
 12-hour nutrient broth cultures of F– E. coli strain L-shaped bent glass 1
thr–, leu–, thi–, and Str-r (ATCC e23724) rod
 Hfr E. coli strain Str-s (ATCC e23740)
1-ml sterile pipettes 3
Media Mechanical pipetting 1

device
Per Lab
Group Per Class Sterile 13-  100-mm 1
test tube
Agar plates of 3
minimal medium plus Glassware marking 1
streptomycin and pencil
thiamine
Waterbath shaker 1

Procedural Points to the National Academy of Sciences,
109(4):1269–74.
Emphasize
1. It is suggested that students be introduced to the
following:
1. Genetic variations are introduced into
a. Genetic mapping and the use of specific
eukaryotic cells by the recombination of
marker genes for the demonstration of the
homologous chromosomes during fertilization
transfer of genetic material between conjugating
and by the process of crossing over. In
cells.
prokaryotic cells, genetic variations occur as a
b. The media that are designed to ascertain the result of conjugation, transformation, and
occurrence of conjugation. transduction.
2. Indicate to the students that the fertility (F) factor 2. The F factor is a small segment of DNA that
contains genes that code for the pilus proteins, allows the cell to serve as the genetic donor.
genes for plasmid replication, and genes required
3. If the F factor is extrachromosomal, a plasmid,
for plasmid transfer.
generally only this factor is transferred.
3. Students should understand that they must However, if the F factor is integrated into the
exercise care when handling cultures and that it bacterial chromosome, designated as Hfr, there
is imperative that the antibiotic-resistant strain is a transfer of a portion of the chromosome.
and plasmids not be spread around the lab.
4. The wild type, Hfr, parental strain is sensitive to
4. Accidental spills or breakage of culture tubes are streptomycin and therefore its growth is inhibited
to be flooded quickly with disinfectant, covered by the streptomycin in the minimal medium. The
with paper towels, and allowed to stand for 30 mutant F– streptomycin-resistant organisms are
minutes. The instructor should be notified when not inhibited by this antibiotic in the medium. As
spills occur. the distance of the Str gene from the thr and leu
genes on the parental chromosomes is too great
for its genetic transfer during conjugation, its sole
Additional Reading function is to serve as a genetic marker. Therefore,
 Stecher, B., Denzler, R., Maier, L., Bernet, F., if growth is present on the minimal medium, it
Sanders, M. J., Pickard, D. J., …Hardt, W. D. would indicate the transfer of the thr+ and the leu+
(2012). Gut inflammation can boost horizontal genes to the mutant F–, streptomycin-resistant
gene transfer between pathogenic and (Str-r) organisms.
commensal Enterobacteriaceae. Proceedings of

EXPERIMENT 54
Isolation of a Streptomycin-Resistant Mutant
The purpose of this experiment is to illustrate the use Equipment

of the gradient-plate technique for the isolation of
antibiotic-resistant mutants. Per Lab
Group Per Class
Material Sterile Petri dish 1

(15  100 mm)
Culture
24-hour nutrient broth culture of: Sterile 1-ml pipettes 2
 E. coli
device
Media
L-shaped bent glass 1
Per Lab rod
Group Per Class
Beaker with 70% 1
10-ml Trypticase soy 2 ethanol
agar deep tubes
Waterbath 1
Inoculating loop 1
Reagent
 Stock streptomycin solution (10 mg per 100 ml Glassware marking 1
of sterile distilled water) pencil

Procedural Points to 2. The gradient-plate preparation establishes an
antibiotic gradient that serves to expose the
Emphasize organisms to differing concentrations of the
1. A demonstration of a gradient-plate preparation antibiotic. In this way, knowledge of the minimal
and an explanation of the resultant antibiotic inhibitory concentration of the antibiotic is not
concentration gradient should be presented. required.
2. Students should be informed that the first layer 3. The extensive use of this antibiotic has served to
of agar medium must be completely hardened select for the drug-resistant bacterial strains by
before pouring the upper antibiotic-containing its bactericidal effect on the sensitive forms. As
layer of agar. a result, there has been an increase in the
streptomycin-resistant microbial population.
3. Spontaneous mutations develop by chance in the
environment and may occur at the rate of one in 4. No. The genetic capability for streptomycin
1 billion replications of the organism. Emphasize resistance was present prior to the advent of this
that resistant mutants will be detected in the antibiotic. The use of streptomycin has rapidly
high antibiotic concentrations in the gradient resulted in the drug-resistant strains because of
plate. the one-step mechanism involved in the
development of streptomycin resistance to all
concentrations of this antibiotic.
Additional Reading
 Liu, Y., Li, J., Du, J., Hu, M., Bai, H., Qi, J., …
Gao, P. (2011). Accurate assessment of antibiotic
susceptibility and screening resistant strains of a
bacterial population by linear gradient plate.
Science China Life Sciences, 54(10):953–60.

1. The mechanisms responsible for antibiotic
resistance include the following: the enzymatic
alteration of the chemical structure of the
antibiotic, a change in cell membrane
permeability, a decrease in microbial enzyme
sensitivity to the inhibitory effect of the
antibiotic, and the overproduction of naturally
occurring metabolites that compete with the drug
for the active site of an enzyme.

EXPERIMENT 55
The Ames Test: A Bacterial Test System

for Chemical Carcinogenicity
The purpose of this test procedure is to demonstrate a Equipment

screening method for the detection of chemical
carcinogens. The Ames test employs bacterial Per Lab
systems, rather than eukaryotic cells, thereby Group Per Class
allowing for the rapid identification of possible
carcinogens resulting from the mutagenic effect of 1-ml sterile pipettes 2
the test agent.
device
Materials
Sterile paper discs as needed
Culture
24-hour nutrient broth culture of: Forceps 1
 S. typhimurium, strain TA 1538 (ATCC e 29631)

Waterbath 1
Media Glassware marking 1

pencil
Per Lab
Group Per Class Disposable gloves 1 pair/
student
Minimal agar plates 4
2-ml top agar tubes 4 Procedural Points to

Emphasize
1. The correlation between mutagenicity and
Reagents
carcinogenicity, which is the basis of this test
 Sterile biotin-histidine solution procedure, should be discussed.
 2-nitrofluorene dissolved in ethyl alcohol
2. Students should be told that the strain of S.
 Two commercial hair dyes
typhimurium selected for this experiment lacks
the genetic information to synthesize DNA repair
enzymes needed for correction of injured DNA.
3. The importance of the incorporation of
mammalian liver enzyme homogenates along
with the chemical agents should be stressed.
Liver enzymes are thought to convert
noncarcinogenic materials to carcinogenic ones.

4. Students must be cautioned to wear gloves and need for metabolism and correlation with rodent
exercise care when handling the test chemicals carcinogenicity. Mutagenesis, 24(4):359–66.
because of their potential carcinogenicity.
5. Students must be given instructions regarding the Answers to Review Questions
disposal of chemically contaminated experimental
materials. Culture plates, prior to autoclaving, 1. The S-9 mix is a liver homogenate that serves as a
must be placed into a “red bag” for identification source of activating enzymes to make bacterial
as hazardous substances. Excess test chemicals and mammalian test systems more compatible.
must be placed into a sealable container for 2. The biotin serves as a bacterial growth
disposal as hazardous wastes, according to the stimulator. The histidine is used to allow the his–
policies prescribed by the institution. organisms to grow, thereby allowing the cells to
undergo cell division, which is necessary for the
mutation to occur.
Optional Procedural Additions
or Modifications 3. Chemical carcinogens cause malignant
transformations in normal tissues by inducing
Other chemical compounds that may be carcinogenic mutations in somatic cells. Therefore, a strong
and of investigative interest to the students can be correlation exists between mutagenicity and
substituted for the suggested test chemicals used in carcinogenicity.
this experiment.
4. The advantages of using bacterial systems to test
for chemical carcinogenicity include the rapid
Additional Reading determination of results and the test procedures’
greater simplicity and lower cost.
 Kamber, M., Flückiger-Isler, S., Engelhardt, G.,
Jaeckh, R., & Zeiger, E. (2009). Comparison of 5. The major disadvantage of using bacterial
the Ames II and traditional Ames test responses systems to test for chemical carcinogenicity is
with respect to mutagenicity, strain specificities, that mammalian and bacterial enzyme systems
are different, and results are not comparable.

EXPERIMENT 56
Bacterial Transformation
The purpose of this experiment is to demonstrate the Plasmid

transformation of a competent antibiotic-sensitive
organism into one that is antibiotic resistant via the Per Lab
insertion of a DNA plasmid. A color marker gene, Group Per Class
which codes for the enzyme beta-galactosidase, is
able to cleave 5-bromo-4-chloro-3-indolyl-ß-D- pBLU 5437 bp long 1
galactoside (called X-Gal) if present in the cell and w/gene for ß-
will produce blue coloration in the transformed cells. lactamase and ß-
galactosidase
Materials
Cultures Equipment
 18- to 24-hour Luria-Bertani (LB) cultures of Per Lab
E. coli (Carolina Biological Supply) Group Per Class
Sterile 13-  100-mm 2

Media test tubes
Per Lab Adjustable 13

Group Per Class micropipette w/sterile
tips or 1-ml
Luria-Bertani agar 4 graduated transfer
base plates pipettes
Luria-Bertani agar 3 Glass beads (6 mm 60

base plates plus in diameter)
ampicillin
Glassware marking 1
Luria-Bertani agar 3 pencil
base plates plus
ampicillin/X-Gal Disposable plastic as needed
inoculating loops
Luria-Bertani broth 1
tube Bunsen burner 1
Waterbath 1
Reagents
500-ml beaker with 1
 50 mM of CaCl2 solution crushed ice
 1.0% ampicillin solution

Equipment (continued) 7. Abrupt heat shock (Step 10) is a critical step.
Transformation tubes should be taken from the
Per Lab ice bucket and transferred to the 42°C waterbath
Group Per Class and then quickly back to the ice.
8. Transformed cells should be spread rapidly. If
500-ml beaker with 1
the cell suspension sits too long on the surface of
disinfectant
the agar plate, too much of the suspension will be
L-shaped bent glass 1
absorbed in one spot, and the cells will not spread
rod (optional) evenly on the plates.
Beaker with 95% 1 Optional Procedural Additions

ethyl alcohol
(optional) or Modifications
Rather than use the glass-bead technique as described
Turntable, if using 1
in the procedure, the instructor may opt to use the
spread-plate method
(optional)
spread-plate technique shown in Figure 2.3 for
plating the cultures.
Wire inoculating loop 1
(optional) Tip
Quebec colony 1  The molarity and pH of CaCl2 is critical for the
counter production of competent cells.

Emphasize  Martínez, J. L. (2011). Bottlenecks in the
transferability of antibiotic resistance from
1. Some strains of E. coli have been associated with natural ecosystems to human bacterial
disease outbreaks, and students may be pathogens. Frontiers in Microbiology, 2:265.
concerned about individual safety. The strains of
E. coli used in molecular biology laboratories do
not contain disease-producing genes and are Answers to Review Questions
harmless under normal conditions. 1. Considering that a DNA plasmid is used in this
2. At the end of the experiment, students must experiment, care must be taken to prevent it from
collect all culture materials and equipment that being introduced into other competent laboratory
have come in contact with the culture and stock cultures of E. coli. Immediate autoclaving
autoclave at 121°C for 15 minutes before of all experimental materials and cultures will
disposal. prevent the possibility of cross contamination.
3. Spilled cultures may be disinfected with a 10% 2. Scientists have the ability to alter the genome of
bleach solution or the disinfectant used in the an organism by transferring the genetic material
laboratory. DNA from one cell to another. There are several
methods, which include transformation,
4. Scraping the agar must be avoided when transduction, and conjugation. Bacterial cells can
transferring the large cell mass, and the cell mass be genetically engineered today to produce
must be deposited directly into the CaCl2 insulin by inserting genes for insulin production
solution and not left on the loop or the side of the into the organism’s genome. Similarly,
tube. interferons can be used in cancer chemotherapy,
5. Cells should not be allowed to clump. Be certain in the treatment of multiple sclerosis, and in the
to suspend the cells rapidly once they are placed production of human growth hormone.
in the CaCl2. Clumped cells are difficult to
resuspend.
6. The DNA plasmid must be placed directly into
the cell suspension.

3. a. The presence of the blue colonies indicates cells begin to reproduce and form cells with new
that the cells have been transformed. The few cell walls, forming satellites in the ampicillin-
white colonies scattered around the blue ones are free zone.
designated as “satellites,” colonies of
b. The absence of growth on these plates
nontransformed cells, growing in an ampicillin-
indicates that transformation did not occur and
free zone. An enzyme that breaks down
the organisms are sensitive to ampicillin.
ampicillin is secreted by the cell and released
into the surrounding medium. The transformed

EXPERIMENT 57
Isolation of Bacterial Plasmids
The purpose of this experiment is to demonstrate the Equipment

isolation of bacterial plasmids. The isolation
procedure commonly used is shown in Figure 57.3. Per Lab
Once the plasmids have been isolated, the students Group Per Class
will separate, visualize, and determine the size of the
plasmid. This is accomplished by the migration of the Microcentrifuge 1–2
plasmid through an agarose gel electrophoresis. The
similarities and differences of each plasmid will be Microcentrifuge tube, 1
determined by comparison of the distance traveled by 2 ml
the separated bands that appear in the gel following
electrophoresis. Micropipettes 10, 1 each
100, 200 μl
Materials Micropipette tips, 1 box each

small and large
Cultures
 24-hour Luria-Bertani broth plus 50 µg/ml of Rubber 1
plasmid-bearing E. coli ATCC 39991 and E. microcentrifuge
tube rack
coli ATCC 51300
Glassware marking 1
Reagents pencil
 Glucose-Tris-EDTA buffer Waterbath 55°C 1

 Tris-EDTA buffer
 Tris-acetate-EDTA buffer Agarose gel casting *
 5 M potassium acetate (KOAc) tray
 Sodium hydroxide containing 1% sodium
dodecyl sulfate (NaOH/SDS) 500-ml beaker 1
 95% cold ethanol w/crushed ice
 70% ethanol
 Molten agarose (55˚C) Plastic sandwich-size 1
 Gel electrophoresis running dye bag
 Carolina Blu stain or 0.025% methylene blue
 HindIII cut bacteriophage lambda DNA Electrophoretic 1
apparatus

Equipment (continued) Optional Procedural Additions
Per Lab
or Modifications
Group Per Class 1. In place of Carolina Blu for staining the agarose
gel, 0.025% methylene blue solution may be
Light box or 1–2 used.
overhead projector
2. Kits for this and other biotechnology
Staining tray 1 experiments are available and eliminate the need
for preparing a variety of reagents and the
Millimeter rule 1 purchasing of different organisms. Kits for the
isolation of plasmids, DNA fingerprinting, and
*The number of casting trays depends on the type of mapping restriction sites on plasmid DNA may
electrophoretic apparatus and the number of wells in be purchased from companies such as Carolina
the agarose gel. Biological Supply Company and Edvotek.

Emphasize  Carattoli, A. (2011). Plasmids in Gram
1. Prior to the lab, demonstrate the proper use of the negatives: Molecular typing of resistance
micropipette and the microcentrifuge, and allow plasmids. International Journal of Medical
the students to practice. Microbiology, 301(8):654–8.
2. One day before the lab, culture the organisms.

Prior to the lab, label a sufficient number of Answers to Review Questions
microcentrifuge tubes EC-1 and EC-2 to 1. Plasmid DNA is preferred over chromosomal
accommodate the number of student groups in DNA for genetic engineering studies because
your lab. Dispense 1 ml of culture to the plasmids are small in size compared to
respectively labeled tubes and use the chromosomes, and they can be easily located and
microcentrifuge. Discard the supernatant and separated.
repeat the procedure. Half the class should obtain
a tube labeled EC-1 and the other half should 2. Plasmids usually carry genes that serve as
obtain EC-2 tubes. selectable markers, such as those that code for
resistance to one or more antibiotics and can
3. During the procedure, caution students to take confer this antibiotic resistance to their bacterial
care in loading the wells in the agarose gel. Hold hosts, thus making plasmids clinically important.
the small-tipped micropipette with both hands
directly over the well. Dip the tip of the pipette 3. a. The EDTA in the buffer chelates the divalent
slightly through the buffer with the tip barely into metal ions, Ca++ and Mg++, which destabilizes the
the well. Discharge the pipette slowly. cell membrane and inhibits the activity of DNases.
The glucose maintains the osmolarity, preventing
4. Be sure that the well-forming comb is seated the buffer from bursting the cell.
properly in the casting tray.
b. SDS is a highly alkaline solution that lyses
5. When removing the well-forming comb from the the cell, releasing the cytoplasm into the buffer,
solidified gel, gently pull it straight up, using and it separates the chromosomal DNA into
care not to damage or tear the wells. single strands (ssDNA) and complexes with
6. Connect the correct electrical leads from the cellular protein.
electrophoresis box to the power supply. c. The KOAc promotes the precipitation of the
7. Exercise care when removing the stained gel chromosomal ssDNA and ssDNA and large
from the casting tray. RNA molecules, which are insoluble in this salt.

4. Alcohol is widely used in molecular biology and cannot be calculated. Because of the difference
is of value because alcohols allow nucleic acids in the structure, the migration of the plasmid and
to concentrate and be precipitated from solution that of the standard cannot be compared directly.
as an insoluble pellet.
7. a. Circular, linear, supercoiled, and nicked.
5. Circular nicked DNA is sometimes referred to as
b. Circular, supercoiled molecules move the
“relaxed” because some of the tension present in
fastest and farthest because they have a very
covalently coiled and twisted DNA has been
compact shape. However, a variety of factors in
released.
any electrophoresis experiment, including the
6. The standard curve describes the migration of type of buffer used, determine the speed and
linear DNA. If the plasmid isolated in an distance of migration.
experiment is circular, the size of the plasmid

EXPERIMENT 58
Restriction Analysis and Electrophoretic

Separation of Bacteriophage Lambda DNA
Restriction endonucleases are enzymes that cleave the Equipment

sugar–phosphate backbone of DNA. In most practical
settings, a given enzyme cuts both strands of the Per Lab
double-helical DNA within a stretch of just a few Group Per Class
bases. A majority of restriction enzymes have been
isolated from bacteria, where they appear to serve a Microcentrifuges 2–3
host-defense role. For example, if a virus infects a
bacterial cell, the foreign viral DNA will be digested Microcentrifuge 5
or inactivated (restricted) within the bacterial host. In tubes, 1.5 ml
this scenario, one finds that the genomic DNA is
spared because the bacterium synthesizing the specific Micropipettes 10, 50, 1 each
endonuclease also synthesizes a DNA 100 μl
methyltransferase, which methylates the selected DNA
sequence and prevents its cleavage. The purpose of this Micropipette tips: fine as needed
experiment is to demonstrate the process of cleaving point, small, and
large
bacteriophage lambda DNA with endonucleases and
separating them using gel electrophoresis.
Waterbath 1–2
Materials Ice bucket w/crushed 1

ice
DNA Source
Staining tray 1
 Bacteriophage lambda (200 μl) (Carolina
Biological Supply catalog number 21-412)
Disposable gloves as needed
Restriction Endonucleases Glassware marking 1

pencil
 EcoRI (21-1607)
 HindHIII (21-1690) Bunsen burner or hot 1
 BamHI (21-1670) plate
250-ml Erlenmeyer 1
Reagents flask
 Tris-acetate buffer
 Type-specific buffers for EcoRI, HindIII, and 500-ml beaker 1
BamHI. See Appendix 4 for preparation
instructions. Floatable foam test 1
tube rack if available
 Electrophoresis loading dye
 Carolina Blu stain or 0.025% methylene blue
 0.8% agarose in IX TAE buffer

Equipment (continued) Additional Reading
Per Lab  Hou, W. R., Xie, S. N., Wang, H. J., Su, Y. Y.,
Group Per Class Lu, J. L., Li, L. L., …Xiang, M. (2011).
Intramuscular delivery of a naked DNA plasmid
Electrophoresis 1–2 encoding proinsulin and pancreatic regenerating
apparatus III protein ameliorates type 1 diabetes mellitus.
Pharmacological Research, 63(4):320–7.
Light box or 1
overhead projector
Millimeter rule 1 1. Restriction enzymes are isolated from bacterial
cells. The temperature environment for bacterial
cells is typically 37°C, which means that these
Procedural Points to enzymes function maximally at this temperature.
Emphasize 2. No, the endonucleases produced by a particular
1. Maintain all restriction enzyme solutions in an bacterial cell will only cleave its specific
ice bucket. sequence on the DNA molecule.
2. Add reagents to the digestion tubes in the 3. Enzymatic reactions are affected by physical
following order: factors such as temperature, specific ionic
strength of the buffer, pH, and the magnesium
a. Lambda DNA ion concentration of the buffer. Each
b. Water endonuclease requires its own buffer to maintain
optimum activity.
c. Buffer
4. Low restriction enzyme activity may be caused
d. Restriction endonucleases by the deterioration of the buffer. This situation
Note: Restriction enzymes must be added to the can be reversed by the use of fresh buffer.
tubes last. 5. The dilution of the enzymes with reaction buffer
3. Review and demonstrate the proper use and care before the addition of DNA might partially or
of the micropipette and allow the students to completely inactivate endonucleases, especially
practice. if the reaction buffer has low ionic strength and
contains no stabilizing agent.
4. Stress that the pipette must be removed
completely from the gel before releasing the 6. Using restriction enzymes, we can cut DNA from
plunger. each of the organisms, ligate them together with
DNA ligase, and insert the ampR and luciferinase
5. Caution students to remove the well-forming gene into the plasmid backbone.
comb from the gel by raising it straight upward
above the gel. 7. We could use restriction enzymes to cut the
human white blood cell interferon and ligate it
6. Stress the importance of attaching the electrical into a plasmid with a gene that is resistant to
leads correctly to the electrophoretic apparatus. ampicillin and then transform the bacterium with
7. Use this as an opportunity to demonstrate the this plasmid. Then all of the ampicillin-resistant
proper use of a microcentrifuge. bacteria growing in a continuous culture will
make that interferon, which can be used to treat
diseases such as cancer, multiple sclerosis, and
Optional Procedural Additions others.
or Modifications
The cost and labor for the preparation of the buffers
and other reagents might be offset by using
commercially prepared kits. Kits for teaching are
available from companies such as Carolina
Biological Supply Company, Ward’s Natural
Science, Fermentas Life Sciences, and Edvotek.

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Microbiology A Laboratory Manual 11th Edition Cappuccino Solutions Manual 1

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Solution Manual for Microbiology A Laboratory Manual

11th Edition Cappuccino Chad 1292176016

60 Copyright © 2017 Pearson Education, Inc.

These experiments are presented to give students a

Reagent G. intestinalis: cyst 1

 Methyl cellulose B. coli: trophozoite 1

Microscope 1 P. vivax (prepared with 1

Pasteur pipette 1 Per Lab

Immersion oil as needed

Copyright © 2017 Pearson Education, Inc. Experiments 32 and 33 61

1. The major distinguishing characteristic between

Parasitic Protozoa Parasitic Protozoa

62 Copyright © 2017 Pearson Education, Inc.

Cultivation and Morphology of Molds

The purpose of these mycological experiments is to Equipment

Cultivation and Morphology of Concave glass slides 4

Cultures Petroleum jelly as needed

Sterile Pasteur pipette 1

Sabouraud agar 1 Inoculating loop 1

Beaker with 95% ethyl 1

Copyright © 2017 Pearson Education, Inc. Experiments 32 and 33 63

Per Lab Coverslips 10

64 Experiments 34, 35, and 36 Copyright © 2017 Pearson Education, Inc.

Glass microscope 1  Glucose–acetate agar is one of the media used to

Copyright © 2017 Pearson Education, Inc. Experiments 34, 35, and 36 65

66 Experiments 34, 35, and 36 Copyright © 2017 Pearson Education, Inc.

Cultivation and Enumeration of Bacteriophages

The purpose of this experiment is twofold. First, it Equipment

Media Test tube rack 1

Per Lab Glassware marking 1

Tryptone agar plates 5

Copyright © 2017 Pearson Education, Inc. Experiments 34, 35, and 36 67

68 Copyright © 2017 Pearson Education, Inc.

Isolation of Coliphages from Raw Sewage

This experimental procedure is designed to Equipment

5-ml tube of 1 1-ml sterile 1

Per Lab Mechanical pipetting 1

Tryptone agar plates 5 Glassware marking 1

Copyright © 2017 Pearson Education, Inc. Experiment 37 69

Disposable gloves 1 pair/ Additional Reading

70 Copyright © 2017 Pearson Education, Inc.

Propagation of Isolated Bacteriophage Cultures

This experimental procedure is designed to Equipment

Materials 1.5-ml centrifuge 1

Per Lab Mechanical pipetting 1

5-ml tube Tris Buffered 1 Micropipette with tips 1

Copyright © 2017 Pearson Education, Inc. Experiment 38 71

72 Copyright © 2017 Pearson Education, Inc.

Physical Agents of Control: Moist Heat

The purpose of these experiments is twofold. First, Media

Sterile test tube 4

Copyright © 2017 Pearson Education, Inc. Experiment 39 73

Cultures Students should be told that Halobacterium

Media Electromagnetic Radiations

Microincinerator or 1 Optional Procedural Additions

74 Experiments 40 and 41 Copyright © 2017 Pearson Education, Inc.

Copyright © 2017 Pearson Education, Inc. Experiments 40 and 41 75

Chemical Agents of Control:

Both of the methodologies presented in this

Materials Per Lab

Mueller-Hinton agar 7 Sulfisoxazole, 150 μg 2

76 Experiments 40 and 41 Copyright © 2017 Pearson Education, Inc.

Forceps 1 PART B: Synergistic Effects of Drug