Week 6





Lab Study Guide

Exercise 14:  Use of Selective & Differential Media & Replica Plating Techniques
1. Define these terms:
a. Complex media – substance containing the nutrients required for microbial growth, but in crude form, meaning their exact quantities are unknown (eg. Nutrient Agar & Blood Heart Agar)
b. Defined media  – substance containing required nutrients for microbial growth, in relatively pure chemical form & exact types and amounts known.
c. Enriched media (also list examples) – Media containing cells, tissue or tissue fractions (Blood Agar & Chocolate Agar)
d. Differential media – media supporting the growth of various types of organisms while distinguishing between different forms of organisms (MAC, MSA, T-7)
e. Selective media – media that supports the growth of specific organisms and inhibiting the growth of others. (MAC, T-7)
2. Explain the differential and selective characteristics of each media shown below.  Know which
pH indicator is used in each media and how to interpret the results (see table called “Selective
& Differential Media”):
a. Mannitol Salt Agar (MSA) – Selective media for isolation of pathogenic & non-pathogenic Staphylococci. (Many other organisms are inhibited by the 7.5% salt content). It is also a differential on basis of mannitol fermentation. pH indicator =phenol red.

Yellow = acidic Pink = neutral   Purple = alkaline

b. MacConkey Agar (MAC) – Selective medium (based on chemicals) for Gram-Negative organisms, inhibiting growth of Gram-Positive. Differential as well for those that can ferment lactose from those that cannot. pH indicator = neutral red. (+) organisms will accumulate red in or around their colonies. (ferment lactose)
c. Eosin Methylene Blue Agar (EMB) – Selective Media (based on chemicals) for growth of Gram-negative bacilli. Differentiates between lactose fermenting colonies and non-lactose fermenting. Lactose fermenting colonies will be a dark/irridescent green color. (+)
d. Tergitol 7 Agar (T-7) – Selective media (based on chemicals) for E. coli & other members of coliform group. Inhibits gram-negative spore-formers and Gram-positive organisms. pH indicator = Bromothymol blue

(lactose fermenter) Yellow = acidicGreen = neutral →  Blue = alkaline (non-lactose fermenter)
3. Explain the function (e.g. benefits) of replica plating. Technique allowing for rapid, efficient transfer of multiple colonies all at one time.
4. What type of material (cloth) was used for obtaining bacteria from the master plate? Why is
this material useful for obtaining bacteria? Velvet; the tiny hair-like projections of velvet readily pick up bacterial cells.
Exercise 15, Supplement B
1. Define dichotomous key.
2. Know how to use dichotomous keys.

Exercise 15-A, B, & C:  Physiological Tests
1. Know the following information about each of the physiological tests (see tables called “Tests
for Gram-positive Organisms” and “Tests for Gram-negative Organisms”):
a. Purpose (question asked with test)
b. Key ingredients, including pH indicator
c. Possible outcomes (results)
d. How to interpret each possible outcome (conclusions)

Oxidase Test
Catalase Activity Test
O/F Test
Simmon’s Citrate Agar
Urea Agar Test
MR-VP Test
Lysine Test
Coagulase Test
Esculin Test
(TSI) Triple Sugar Iron Test

  • Q: Does organism ferment more than 1 sugar?
  • Q: Does the organism produce H2S (Hydrogen Sulfide)?
  • Ingredients: Glucose, Sucrose, Lactose
  • Ingredients: Iron
  • (+): Red slant/yellow butt = organism ferments glucose only; or yellow slant/yellow butt = organism ferments more than 1 sugar (crack = gas fermentation)
  • (+):black agar= organism produces H2S
  • (-): red slant/red butt = does not ferment any sugar
  • (-): no black = no H2S production.

(SIM) Sulfur Indole Motility Test

  • Q: Is organism motile?
  • Q: Does organism produce indole from catabolism of tryptophan?
  • Q: Does organism produce H2S?
  • Ingredient: Low concentration of agar
  • Indgredient: Tryptophan + Kovac’s Reagent (day 2)
  • Ingredient: Iron, forming iron sulfide precipitant
  • (+): “cloudiness” away from stab = motile
  • (+): Kovac’s forms cherry red color =  organism produces indole during catabolism of tryptophan.
  • (+): Black = organism produces H2S
  • (-) results are opposite of positive, (no cloudiness, nor bright red, not black)
  • Procedure: Using NEEDLE, make straight stab to bottom of Agar tube.

Carbohydrate Utilization Test 

  • Q: Does this organism catabolize _______________?
  • Ingredients: Select Carbohydrate
  • pH indicator: phenol red
  • (+): Turns media yellow
  • (-): Stays red/pink
  • Procedure: use loop & stab to bottom of tube.

2. What are enteric bacteria?  Be able to recognize these enteric bacteria on the list of organisms
for our physiological unknown #1 samples (see table):
Citrobacter fruendii  Enterobacter aerongenes
Enterobacter cloacae  Escherichia coli
Klebsiella oxytoca   Klebsiella pneumonia
Proteus mirabilis   Proteus vulgaris
Salmonella choleraesuis  Serratia marcescens
Serratia odorifera

Note that these enterics are Gram-negative rods and also O-F positive (fermentative)

3. Be able to recognize the Gram-positive bacteria on the list of organisms for our physiological
unknown #1 (see table):
Bacillus cereus   Bacillus sphaericus
Bacillus subtilis   Enterococcus faecalis
Staphylococcus aureus  Staphylococcus epidermidis
Streptococcus mitis  Streptococcus pyogenes

Lecture Study Guide

Microbial Metabolism (Chapter 5)
1. Define the terms metabolism, anabolism, and catabolism.
2. Define the terms oxidation and reduction.
3. List the three ways that ATP is formed.
4. What are enzymes?
5. Explain how enzymes increase the rate of a chemical reaction.
6. Define the terms apoenzyme, holoenzyme, and coenzyme.
7. Be able to recognize examples of inorganic cofactors and coenzymes.
8. Define the terms active site and substrate.  Be able to recognize these items on a diagram of
an enzyme.
9. What is protein denaturation?  What factors cause protein denaturation?
10. Explain how feedback inhibition works.
11. Explain the difference between a competitive enzyme inhibitor and a noncompetitive enzyme
12. Define carbohydrate catabolism.
13. List two general differences between cellular respiration and fermentation.
14. Define aerobic respiration.
15. Define anaerobic respiration.
16. Write the general equation for aerobic cellular respiration of glucose.
17. List the 4 stages of aerobic cellular respiration (including the acetylCoA formation step).
18. Explain what happens during each stage of aerobic cellular respiration.  Example:  During
glycolysis, glucose is broken down into two 3-carbon molecules called pyruvic acid
19. Explain the role of nicotinamide adenine dinucleotide (NAD+) and flavin adenine dinucleotide
(FAD) during the Krebs cycle.
20. Know how many molecules of ATP, CO2 and NADH or FADH2 are produced during each
stage of aerobic cellular respiration.
21. Know where each stage of aerobic cellular respiration occurs inside eukaryotic cells vs.
prokaryotic cells.
22. Be able to recognize reactions that occur during the preparatory (energy investment) stage of
glycolysis vs. reactions that occur during the energy-conserving (energy payoff) stage of
23. Explain what happens to protons and electrons during the electron transport chain (also, what
are the sources of the protons and electrons?).
24. Define chemiosmosis and explain its role in the formation of ATP.

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