Exercises 7 -12
Biol 2230 Lab Questions
Questions For Exercise 7
1. Give the optimum temperature range for the following:
a. psychrophiles - 00C 200C
b. mesophiles - 250 C 400 C
c. thermophiles - 400 C 1000 C
d. psychrotrophs - 200 C 300 C
2. Which of the groups listed in #1 would:
a. grow on the ocean floor - psychrophiles
b. grow in geysers - thermophiles
c. spoil food in the refrigerator - psychrotrophs
d. cause disease in animals - mesophiles
3. Will members of any of these groups (#1) grow in your freezer? no
4. Will members of any of these groups (#1) survive in your freezer? yes
5. What is the term for a microbe that grows at 50 C, 220C and 370C with
optimum growth at 220C? psychrotrophs
6. Define the following terms:
a. acidophiles microbes that grow optimally below pH 6.0
b. neutrophiles microbes that grow optimally at pH 6.0 8.0
c. alkalophiles microbes that grow optimally at a pH above 8
7. To which of the three groups (#6) do molds and yeasts belong? acidophiles
8. How do extremes in pH inhibit microbial growth?
Inhibit enzyme activity, metabolism and growth
9. What is the function of buffers? maintain constant pH
10. Define the following terms:
a. strict aerobe microbes requiring atmospheric level (21%) of free O2 for
growth
b. microaerophile microbes requiring less than 21% free O2 for optimal
growth
c. facultative anaerobe microbes that use free O2 when available but can
also grow in its absence
d. obligate anaerobe microbes that cannot grow in presence of free O2
e. aerotolerant anaerobe microbes that grow equally well in presence or
absence of free O2
11. In which of these groups (#10) can the Pasteur effect be observed?
facultative anaerobes
12. What is the function of thioglycollic acid in thioglycollate medium?
reducing agent that removes oxygen
13. How can the Pasteur effect be identified in the thioglycollate medium?
heavier growth at top of medium where there is some free O2 and lighter
growth below surface where there is no free O2
14. Which of the groups in #10 will grow both in the anaerobic jar and in
thioglycollate medium? facultative anaerobes, obligate anaerobes and
aerotolerant anaerobes
15. Which of the groups in #10 will grow both in thioglycollate medium and on
the aerobic plate? facultative anaerobes and aerotolerant anaerobes
16. Which of the groups in #10 will grow only on the aerobic plate?
obligate aerobes
17. Why is methylene blue added to thioglycollate medium?
It is a reducible dye that is blue when oxidized showing the presence of some
O2 at the top of the medium
.
18. How is the oxygen removed in the anaerobic jar?
chemical reaction produces CO2 & H2 gas that combines with free O2 to form H2O
19. How does oxygen prevent the growth of an obligate anaerobe?
toxic products such as hydrogen peroxide (H2O2) and super oxide ions (O2-)
are formed that cannot be broken down due to the absence of catalase & SOD
Questions For Exercise 8
1. Define the following terms:
a. thermoduric bacteria-
mesophiles that survive exposure to high temperaturesfor short periods of time
b. psychrotroph- mesophiles that can grow at temperatures below 200C and
spoil food in the refrigerator
c. spoilage- any undesirable change in smell, taste or texture of food
d. TFTC- too few to count; less than 30 colonies on a plate in plate count
technique
e. TNTC- too numerous to count; more than 300 colonies on a plate in the plate
count technique
f. plate count- adding diluted samples to agar plates, incubating and counting
colonies produced to determine number of bacteria in the original culture or sample
2. List three factors that affect the number of bacteria present in a food.
type of food, method by which food is processed and method of storage
3. How is a serial dilution performed? A fixed volume is removed from a culture or
sample and added to a tube containing a specific volume of dilution fluid. Then a
fixed volume from that tube is added to another tube of dilution fluid. This transfer
process is repeated until the desired dilution is obtained..
4. List three types of pipets used in Biology labs.
Pasteur pipets, volumetric pipets and graduated pipets
5. Which of the three types can be used to transfer exact volumes within a
fixed range? graduated pipets
6. Using a 1.0 ml graduated pipet to transfer 0.6 ml, the liquid should be drawn
up to which mark? O.4 ml
7. How is using a blow-out pipet different from using a drain-out pipet?
the volume at the tip of a blow-out pipet is transferred but it is discarded when
using a drain-out pipet
8. List two precautions that are used to prevent contamination of sterile pipets.
handling only the upper portion and never allowing the lower portion to touch a
nonsterile surface
9. What dilution is produced when 10 grams of food are added to 90 mls of
saline? 1 to 10 or 10-1
10. What dilution is produced when 1.0 ml of a sample is added to 9.0 mls of
saline? 1 to 10 or 10-1
11. What is the purpose of the serial dilution procedure?
to reduce the number of bacteria so each will form an isolated colony on the plate
12. How does adding 0.1 ml instead of 1.0 ml of a dilution to an agar plate
affect the dilution factor? Increases it by one-tenth (10-2 to 10-3)
13. List four groups of bacteria that are food pathogens.
Clostridium botulinum, some strains of Staphylococcus aureus, some srains of
Escherichia coli and some strains of Salmonella
14. How is processing related to the number of bacteria present in a food?
Cutting, shredding and exposure to air and nonsterile surfaces
increase the number of bacteria in the food.
15. Do the large number of bacteria in milk and hamburger pose a health
hazzard? Explain. No; Pasteurization kills any pathogenic bacteria in milk and
proper cooking kills any pathogens present in the hamburger.
16. Which potential pathogens can be isolated using EMB agar and MSA ?
EMB agar- coliform bacteri such as E. coli
MSA agar Staphylococcus aureus
17. How can spoilage of food be prevented or delayed? by destroying the
microbes during processing or storing food at a temperature that does not
permit growth of bacteria that survive processing
18. What types of bacteria can be isolated from canned foods?
thermophiles and thermoduric bacteria
19. What types of bacteria are found in pasteurized milk?
nonpathogenic psychrophiles and psychrotrophic bacteria
20. Do frozen foods contain a significant number of viable bacteria? yes
21. Would the presence of Clostridium botulinum in a frozen food pose a
health hazard? Explain. No; It cannot grow and produce its toxin in the
freezer and cooking will kill the vegetative cells that produce the toxin.
22. Would the presence of Clostridium botulinum in a can of beans pose a
health hazard? Explain. Yes; Endospores could germinate producing
vegetative cells that can multiply producing its deadly toxin. Then, if the food is
not properly cooked botulism can occur.
23. What is the basic assumption relating the number of colonies to the
number of bacteria in the diluted sample? Each isolated bacterium in a
diluted sample will produce one colony.
24. What is the ideal colony counting range? 30-300 colonies
25. Why are plates with less than 30 colonies designated TFTC?
Contamination and sampling error can have a significant effect on the number
of colonies counted and the number of baceria per gram calculated from the count.
26. If 0.1 ml from a 10-2 dilution of a food sample were added to an agar plate
and produced 90 colonies, what is the number of bacteria per gram in the
food? Express your answer in standard scientific notation.
9.0 X 104 bacteria per gram
Questions For Exercise 9
1. What are enzymes? molecules that act as biological catalysts accelerating
chemical reactions in living cells
2. Define the following terms:
a. exoenzyme - enzyme released by microbes that can break down large
molecules, often nutrient sources, in the environment
b. endoenzyme - enzymes produced by microbes that function inside them
c. substrate - molecule with surface that fits a specific enzyme and can
combine with and be altered by it
d. hydrolysis splitting of molecules by addition of water to them
3. Characterize the following as either an exoenzyme or endoenzyme:
a. amylase - exoenzyme
b. catalase - endoenzyme
c. lipase - exoenzyme
d. oxidase - endoenzyme
e. gelatinase - exoenzyme
4. Starch is a __e___.
a. lipid d. dextrin
b. protein e. polysaccharide
c. disaccharide
5. amylase is the enzyme that hydrolyzes starch.
6. How is starch hydrolysis detected when examining a starch agar plate?
iodine is added & reacts with starch present to produce a purple color leaving a
clear area around microbial growth where starch has been hydrolyzed
7. What is tributyrin? a fat
8. How is hydrolysis of tributyrin detected?
hydrolysis of fat droplets dispersed in the agar results in clearing of agar around the
growth
9. _____lipase______ is the enzyme that hydrolyzes tributyrin.
10. Gelatin is a __b___.
a. lipid d. dextrin
b. protein e. polysaccharide
c. disaccharide
11. Why are tubes of gelatin put in the refrigerator prior to observing for
hydrolysis? Above 250C (in incubnator) gelatin is liquid. Refrigeration solidifies
the undigested gelatin but the hydrolyzed gelatin remains liquid.
12. _______gelatinase______ is the enzyme that hydrolyzes gelatin.
13. Give the end products of the complete hydrolysis of each of the following:
a. starch - many glucose molecules
b. lipid - glycerol and fatty acids
c. protein - amino acids
14. What is the action of catalase?
Breaks down hydrogen peroxide ( H2O2)
15. How is catalase production detected? Oxygen is released forming numerous
bubbles when hydrogen peroxide is added.
16. What is the relationship between oxygen usage and catalase production?
Use of O2 in respiration produces some H2O2 which is toxic. Obligate aerobes
and facultative anaerobes typically produce catalase which permits their
growth in O2. Obligate anaerobes die in free oxygen in part because they lack catalase.
17. List two genera that do not produce catalase.
Streptococcus and
Clostridium
18. What is the function of oxidase in bacteria?
It catalyzes the transfer of electrons to the last cytochrome carrier in the
electron transport system.
19. What is the reagent used to detect oxidase?
reduced tetramethyl-p-phenylenediamine dihydrochloride
20. What color do oxidase-positive colonies turn? purple
21. Is oxidase production more common in aerobic or anaerobic bacteria?
aerobic bacteria
22. List two pathogens that are oxidase-positive.
Pseudomonas aeruginosa and Neisseria gonorrhoeae ( N. meningitides)
Questions For Exercise 10
1. List two major differences between fermentation and respiration.
Respiration is a complete oxidation of organic molecules using an ETS and
producing inorganic products. Fermentation is an incomplete oxidation of
organic molecules that does not use an ETS and often produces organic products.
2. Is fermentation due to exoenzymes or endoenzymes? endoenzymes
3. List three groups of products of fermentation.
acids, gases and alcohols
4. Explain the difference between homofermentative microbes and
heterofermentative microbes. Homofermentative microbes produce only lactic
acid in fermentation. Heterofermentative microbes produce various organic
products that may include lactic acid..
5. Which of the following groups are capable of both fermentation and
respiration?
a. strict aerobes
b. facultative anaerobes
c. obligate anaerobes
d. aerotolerant anaerobes
b. facultative anaerobes and c. obligate anaerobes
6. Which of the groups in #5 do not ferment carbohydrates?
a. strict aerobes
7. What is the function of phenol red in the sugar broths?
pH indicator that detects acid production
8. What is the function of the Durham tube?
collects gas produced in fermentation by some microbes
9. How is the production of acid detected in the sugar broths?
Broth changes from red to yellow
10. What process and product can cause sugar broth to develop a dark
red color? breakdown of proteins releasing ammonia
11. Of what value is the fermentative pattern of a bacterium to the
microbiologist? identification and
classification of different species of bacteria
Questions For Exercise 11
1. What are coliform bacteria? Gram-negative bacilli found in large intestine of
humans &warm-blooded animals
2. What do the letters in IMViC stand for? I - indole production; V - Voges-
Proskauer test; M - methyl red test; i - just for pronunciation; C - citrate utilization
3. How are the IMViC reactions used? To differentiate coliform species in human
colon
4. How are the methyl red and Voges-Proskauer tests related? They detect
different products of glucose fermentation.
5. What is methyl red? pH indicator
6. What products of fermentation are detected by the methyl red test? acids
that lower pH to 4.2 or below
7. What color indicates a positive methyl red test? a negative test?
positive red; negative yellow or orange
8. The Voges-Proskauer test identifies what compound?
acetoin (acetylmethylcarbinol)
9. What reagent is used in the Voges-Proskauer test? Barritts A &B
10. What color change is produced in a positive Voges-Proskauer test?
red color develops
11. What type of medium is Simmon's citrate agar?
chemically defined
12. What exoenzyme is produced by bacteria that can grow on this medium?
citrase
13. What color change indicates citrate utilization? green to royal blue
14. What molecule is responsible for the color change?
sodium carbonate from release of CO2
15. Does the color change in Simmon's citrate agar indicate an acidic, a basic
or a neutral pH? basic
Questions For Exercise 12
1. List two ways in which hydrogen sulfide may be produced by bacteria
growing in SIM medium.
breakdown of sulfur-containing amino acids such as cysteine; reduction of
inorganic thiosufate in the medium
2. What compound is produced by the combination of H2S with ferric ions?
Ferric sulfide (FeS)
3. How is production is H2S recognized in SIM cultures? FeS is black
precipitate
4. What amino acid is broken down to produce indole? tryptophan
5. What reagent is added to test for indole? Kovacs reagent
6. What color change indicates the presence of indole? red color after Kovacs
reagent is added
7. What property of SIM medium allows the identification of motile bacteria?
very low concentration of agar (0.3%) which produces a soft solid medium
8. How is motility detected in SIM agar? growth away from line of inoculation
through medium
9. What is the action of urease? It splits urea.
10. ammonia is the product of urease activity that
changes the pH of urea broth.
11. What color change indicates urease production? medium changes from light
orange to bright pink color
12. What is litmus milk? milk containing the dye , litmus
13. What two nutrients in litmus milk are broken down by bacteria?
lactose which is a disaccharide and casein which is a protein
14. What two properties of litmus allow the identification of the various
reactions in litmus milk? It is a reducible dye (colorless when reduced) & also
a pH indicator
15. Describe the appearance of the following litmus milk reactions, list the
nutrient utilized (broken down), and the products released.
a. litmus reduction white with blue at top; utilization of lactose ;
release of H2
and/or electrons
b. acid curd solid curd with pink color; fermentation of
lactose; acid & in
some species gas also
c. proteolysis translucent(sometimes muddy) with purple ring at
top; rapid
breakdown of casein, ammonia released
d. alkaline reaction medium turns dark purple; slow breakdown of
amino
acids in casein, ammonia released