Microbiology Study Guide—CLASS
SET
Directions: Answer the following questions ON YOUR OWN PAPER and in COMPLETE
SENTENCES. First, let’s go all the way back to the beginning before we look at the results for
the end of the lab series!
Biotechnology Lab Manual (Lab 4e pg. 71-72)
1. What is the food source called when growing bacteria in a lab? What are the two kinds?
2. How do you decide which medium to use when growing bacteria? (be specific)
3. Write out the media prep equation.
4. Use the media prep equation for the following problem. The recipe on the stock bottle
states to use 25 g of media base in 1L but we are only going to prepare 175 mL. How much
media base (in grams) will be needed to make 175 mL? (show your work)
5. Explain how you would prepare the above solution once you have the amount of media base
that is required.
6. Why do you want to prepare your solutions in containers that are at least 2X the volume of
the prepared media volume?
7. Critique your skill at making LB broth. Were there any issues or problems that you
encountered? What was one issue that Mrs. Malone encountered and how was it handled?
Biotechnology Lab Manual (Lab 4f pg. 74-76)
8. When is sterile technique used?
9. How do we sterilize media used in biotechnology?
10. Why is high temperature important for sterility?
11. What is sterile technique? (list examples of what you would do)
12. What should be done BEFORE you begin pouring plates?
13. Name 5 things you can do to decrease the chance of contaminating a sample.
14. When pouring plates, you notice that the agar is coming out in lumps. Why is this
undesirable and what corrective measures can you take?
15. LB agar plates are needed for several experiments. If 9 sleeves of Petri dishes (20 plates /
sleeve) are needed, each poured with 25 mL of agar solution, what total volume of LB agar
should you prepare?
16. Use the media prep equation for the following problem. The recipe on the LB agar stock
bottle states to use 40 g of agar media base in 1L but we are only going to prepare 125 mL.
How much media base (in grams) will be needed to make 125 mL? (show your work)
17. Explain how you would prepare the above solution once you have the amount of media
base that is required.
18. Critique your skill at pouring LB agar plates. Were there any issues or problems that you
encountered? What was one issue that Mrs. Malone encountered and how was it handled?
Microbiology Lab #1/ 3A (Aspetic Technique- Inoculate and Observe LB Broth Cultures)
19. Name the six bacterial species that were studied in this lab series.
20. What is aseptic technique?
21. What is the purpose of the control sample for the LB broth culture tubes? How do you
“inoculate” the control sample?
22. The next day you observe your samples and let’s say you notice that your control sample is
“cloudy”. What does this result tell you?
23. Describe how you inoculate your “sample” tube using bacteria from the stock tube.
24. Where did we put the inoculated broth tubes for optimal bacterial growth?
25. Reflect on your inoculating technique. What were the challenges? Were you successful?
Did the dry run help you master this technique?
26. Go back and look at “your” results (lab 3a). Did you have any contamination? How do you
know? Did your bacterial species grow well in the broth? How do you know?
27. Make a bulleted list of all bacterial species describing how they grew (color/ descriptions of
Growth, etc.)
28. What is a pellicle?
Microbiology Lab #7 (Carbohydrate Fermentation)
29. What was the purpose of lab 7?
30. Cells break down glucose in a process known as _____________________.
31. Anaerobic bacteria produce what as their waste products?
32. For bacteria that are oxidative, the pH change will be visible only at the TOP of the tube
where oxygen is available for metabolism. What might the color look like in this tube?
33. What is the name of the pH indicator that is used in the broth solution?
34. A red color in the tube indicates a pH of ___________. A yellow color in the tube indicated
a pH of _________. Why does the color change from red to yellow? (explain in detail)
35. What is a Durham tube?
36. Fill out the results (note: write in pencil so corrections can be made; we will go over results)
Organism
PR/ Dextrose+ and PR/Lactose + and
Conclusions about
color
color
culture
Bacillus sp.
Staphylococcus
epidermidis
Enterococcus faecalis
Pseudomonas aeruginosa
Escherichia coli
Serratia marcescens
Key for table
A = acid produced – broth changed from red to yellow
G = gas produced – bubbles present (or obvious displacement of solution) in Durham tube
A/G = acid and gas are both produced
NC = no change in color, no gas present
* Make sure to note the color change, if any.
**Also describe if the change in color extends throughout the tube or is only present at the top of the solution.
37. Which bacteria were lactose fermenters? Which bacteria were Dextrose fermenters?
How do you know? (I will give you’re the answers)
38. Were there any bacteria that were able to ferment both types of carbohydrates? (list
them)(I will give you the answer)
39. Why would it be advantageous to be able to ferment different types of sugars?
Microbiology Lab#2 / 3B (Aseptic Streaking and Observation of Agar Plates)
40. What is the “streak plate” method?
41. What is the objective of this method?
42. Draw and explain how to use the “Z streak” method to streak your LB agar plates.
43. a) When would you NOT want to flame your loop before every 1/3 turn when streaking
your LB agar plate?
b) Did you find this was the case when practicing your streaking technique in the lab?
EXPLAIN
44. Go back and look at “your” results (lab 3B). Did you have any contamination on your
plates? How do you know (what would contamination look like)?
45. Make a bulleted list of all bacterial species DESCRIBING how they grew (include the
following information in your list: size of colony; color of colony; surface of colony; and odor of
colony)
Microbiology Lab #6 (Differential vs. Selective Plating)-you will also need to consult your
lecture notes for this section
46.
47.
48.
49.
What was the purpose of this lab?
Name and define the 4 types of media that are used to isolate bacteria (see lecture notes).
What does “enteric” mean?
EMB plates will only grow _______________bacteria due to the dyes and ability to ferment
lactose.
50. EMB plates are selective, differential, or selective & differential. (circle your answer)
51. a) Which bacterial species should be able to grow on these plates (EMB)?
b) Describe what the colonies would look like for strong, weak, and non-lactose
fermenters.
52. CNA blood plates will only grow _______________bacteria because they can grow in the
presence of the antibiotics added to the plates but also due hemolysis of the blood in the
plates.
53. What is the name of the enzyme used to lyse the red blood cells in the plates?
54. CNA is a type of blood agar plate that is both selective and differential. Describe how
these plates are differential.
55. Which bacterial species should be able to grow on these plates (CNA)? Discuss the 3
categories of hemolysis in your answer.
56. MacConkey Agar plates are used to grow _______________ bacteria.
57. The lactose in the MAC plates allows differentiation of these types of bacteria. Describe
what the colonies will look like with strong, weak and non-lactose fermenters.
58. Fill out the results (note: write in pencil so corrections can be made; we will go over
results)
Organism
EMB Agar
Growth
Color of
or
Colonies
No
growth
Lac+
or
Lac-
Growth
or
No
growth
CNA Agar
Color of
Hemolysis
Colonies
and type?
MacConkey Agar
Growth
Color of
Lac
or No
Colonies
+ or
growth
Lac -
Bacillus sp.
Staphylococcus
epidermidis
Enterococcus
faecalis
Pseudomonas
aeruginosa
Escherichia
coli
Serratia
marcescens
59. Make a TABLE of the gram positive and the gram negative bacterial species that we have
been studying.
60. You streak Escherichia coli on a CNA plate. You notice growth on the plate. What can you
conclude about this result?
61. You streak Staphylococcus epidermidis on a CNA plate. You have small white colonies on
the plate with no clear zones around the colonies. What can you conclude about the result?
Discuss hemolysis in your answer.
62. You streak Escherichia coli on a MAC plate. What should you expect to find after growing
the colonies for 48 hours? Describe your expected results IN DETAIL.
Microbiology Lab#4 (Heat Fixing and Staining Methods)
63. What is the purpose of lab 4?
64. Describe how you heat fix the bacteria onto microscope slides?
65. Why did we heat fix the bacteria to the slide? Explain.
66. Did you encounter any problems or challenges when heat fixing your bacteria?
67. What are the two types of dyes or stains? Explain each.
68. Explain the properties of gram negative bacteria. (discuss color and why they stain this
color)
69. Explain the properties of gram positive bacteria. (discuss color and why they stain this
color)
70. Gram staining is differential. What does this mean?
71. Who developed the gram staining method? When?____________________________
72. What are the 4 reagents needed to perform a gram stain? (list names of the actual stain)
73. What is a “mordant”?
74. What is the name of the mordant used for gram staining?____________
75. What was the name of the “decolorinzing agent”? __________________
76. What is the purpose of the decolorizing agent? Explain in detail.
77. Why do they decolorizing is the “most critical step” in gram staining?
78. What is the purpose of a counterstain?
79. What is the name of the counterstain used in gram staining?_______________
80. Which bacteria are considered more pathogenic (toxic) gram positive or gram negative?
Why?
81. A new lab technician started working in your lab. Your job is to teach her how to gram
stain. Please write out the appropriate steps needed so she can properly stain a prepared heat
fixed slide containing a mix culture of bacteria.
82. Discuss any issues or challenges your group faced during gram staining.
Lab 5: Bacterial Morphology and Microscopy
83. What is the purpose of a QC slide during gram staining?
84. What should a properly stained QC slide look like?
85. What are the basic bacterial shapes?
86. What are the most common arrangements that bacteria can form in broth?
87. You look at a gram stained slide under the scope and see small purple bacteria that are
spherical and arranged in chains. Write a description that would properly document the
bacteria.
88. How did looking at our bacteria fixed from broth differ from those same bacteria fixed from
agar plates? (look in background info.)
89. What was one really good tip to take note of when looking at bacilli shaped bacteria under
the scope?
90. What was the total magnification used to view our bacteria? What did we have to add to
the slide in order to view them with our microscope?
91. Describe what the results SHOULD look like for our 6 species when viewed under the
microscope using gram staining (QC slide was perfect!).
92. You viewed E. coli under the scope and found both purple and pink rods. What does this
indicate (what happened-contamination)?
93. You viewed Staphylococcus under the scope and found mainly pink cocci. What does this
indicate?
94. What was the primary documentation used to capture your bacterial observations?
95. Did your group have any difficulties or challenges during this lab? If yes, how were they
overcome?
96. You look at a gram stained slide under the scope and see small pink rod-shaped bacteria
that are arranged in clusters. Write a description that would properly document the bacteria.