Category: Microbiology

Starch hydrolysis.

	Starch Hydrolysis
E. coli	-
E. aerogenes	-
P. aeruginosa	-
S. aureus	-
M. luteus	-
B. cereus	+

 

Reference

Cappuccino, J. G., & Welsh, C. (2018). Microbiology: A laboratory manual.

 

 

 

Casein nutrient agar for casease.

	Milk Agar
E. coli	-
E. aerogenes	-
P. aeruginosa	+
S. aureus	-
M. luteus	+
B. cereus	+

Reference

Cappuccino, J. G., & Welsh, C. (2018). Microbiology: A laboratory manual.

Cultivating anaerobic organisms.

	FTM	-O2	+O2	Oxygen Requirements
M. luteus	Top	-	+	Strict aerobe
B. cereus	Throughout	+	++	Facultative aerobe
C. sporogenes	Below O2 layer	+	-	Strict anaerobe

Reference

Cappuccino, J. G., & Welsh, C. (2018). Microbiology: A laboratory manual.

Differential, selective, and enriched media.

Full-size poster download here.

Selective media.

Isolates or is “selective for” certain groups/types of bacteria by incorporating chemical agents that inhibit the growth of certain organisms and promote the growth of other organisms. Selective media include (but not limited to):

Phenylethyl alcohol agar. Isolates Gram-positive organisms. Phenylethyl alcohol partially inhibits Gram-negative organisms.

Crystal violet agar. Selective for most Gram-negative organisms. Inhibits most Gram-positive organisms.

7.5% sodium chloride agar. Promotes halophilic organisms such as Staphylococcus, and is inhibitory for most others.

Differential/Selective media.

These types of growth media incorporate materials that aid in selection (promote/inhibit growth) and morphological differentiation.  Examples are (but not limited to) MacConkey agar, Mannitol salt agar, and Eosin-methylene blue agar.

MacConkey agar. Contains bile salts and crystal violet which inhibit Gram-positive organisms, but promote the growth of Gram-negative organisms. MacConkey also contains lactose and pH indicator neutral red which distinguishes between lactose-fermentors (red) and non-lactose-fermentors (translucent). Enteric bacteria may be separated into lactose-fermentors and non-lactose-fermentors.

• Coliform bacilli: lactose fermentor which produces an acid by-product. Will appear red. E. coli will also turn the surrounds pink.
• Dysentery, typhoid, and paratyphoid: non lactose-fermentors. Will appear tan or transparent.

Mannitol salt agar. Promotes halophilic organisms (e.g. staphylococci) as this medium contains 7.5% NaCl (and inhibits most but not other organisms). The differential components are: mannitol which some staphylococci can ferment; pH indicator phenol red which detects acid produced from mannitol fermentation (creates a yellow-zone).

Eosin-methylene blue agar (Levine). Helps distinguish between enteric lactose-fermentors and non-lactose-fermentors and colon bacillus (E. coli). E. coli will appear blue-black with a green metallic sheen due to large amounts of acid by-products. E. aerogenes will make a thick mucous-looking pink colonies. Non-lactose-fermentors will appear transparent and unremarkable.

Enriched media.

Enriched media contains generous amounts of certain types of nutrients. For example, blood agar can contain 5% sheep blood to promote growth of fastidious organisms such as Streptococcus spp. Organisms that favor blood agar demonstrate hemolysis (breakdown of heme, blood).

Gamma hemolysis. No lysis of blood; no visible change in the medium.

Alpha hemolysis. Incomplete hemolysis resulting in a greenish halo surrounding the colonies.

Beta hemolysis. Complete hemolysis resulting in a clear zone around the colonies. Streptolysin O produces hemolysis by an antigenic, oxygenlabile enzyme. Streptolysin S is a nonantigenic oxygen-stable lysin.

TSA—tryptic soy agar; MSA—mannitol soy agar; MAC—MacConkey agar; PEA—phenylethyl alcohol agar; EMB—eosin methylene blue agar; BDA—blood agar.

Reference

Cappuccino, J. G., & Welsh, C. (2018). Microbiology: A laboratory manual.

Bacillus subtilis at 1000x

Shaeffer-Fulton Method, infrared contrast filtered. Primary malachite green; secondary safranin.

Reference

Cappuccino, J. G., & Welsh, C. (2018). Microbiology: A laboratory manual.

Serratia marcescens

25 °C —Pigment prodigiosin.

37 °C —No pigmentation.

Reference

Cappuccino, J. G., & Welsh, C. (2018). Microbiology: A laboratory manual.

Designing the Dichotomous Key

When designing a dichotomous key, each level represents 1 step in your logic/questioning. The “di” in the word dichotomous means “two”: yes or no. The key is like an upside down tree. At each level, ask a yes/no question. The “yes” should lead the questioning/logic in one direction/branch; the “no” should lead the questioning/logic towards another direction.

See this example.

How to succeed in your microbiology unknown project.

The most common microbiology project is the correct identification of a bacterium using as few tests/diagnostics as possible. This is usually an “end of quarter” project. Here are some tips (in no particular order):

• The most important tip is to keep your mind open. Let the science speak for itself. Don’t make any assumptions. If you make assumptions from the get-go (e.g. it’s E. coli), then you’ve skewed your perspective and your logic. If you start off looking for something to confirm your assumptions, then you’re going to misinterpret your results.
• Pay attention to your class lab results during the rest of the quarter. Pool data and results from classmates. Learn about the different strains of bacteria you’ve handled. There’s a really good chance that your “unknown” will come from one of the bacterium you’ve previously handled/examined earlier in the quarter.
• If the teacher gave you a list of possible bacteria, then try to eliminate half of the pool (via test results) at every step in your dichotomous key. Create a dichotomous key! For example, the teacher gives you a list of 10 possible bacteria. Separate Gram-positive from Gram-negative. Research all the other qualities of the bacteria and put your research in a matrix chart (examples in pictures below). This matrix will help you create your dichotomous key and help you decide the tests and media you will use.
• During the quarter leading up to the unknown project, take lots of pictures of your results and your classmates’. This will provide a great visual key/reference.
• Develop excellent aseptic techniques as soon as possible.
• Maintain a stock of pure cultures and a working cultures. Label, label, label. I cannot emphasize that enough.
• Create a naming schematic and spreadsheet for your pure/working cultures. The name should tell you which generation of specimen it is. This is an example: 08012018plate. Followed by 08012018plate08072018 (which tells me that I created another plate, 08072018, from the 08012018 plate). Or 08012018plate08072018slant. That tells me that I created a slant, 08072018, from my 08012018 plate. Get the idea? You want to keep track of your generations. And YES…keep the cultures (as many as reasonable), and make sure you have relatively “fresh” batches to do your testing. If you’re 16 days into your unknown project, you’re NOT going to want to use the original plate.

Greetings! Feel free to download (click on the thumbnails to get the PDFs) these lab forms if you find them to be helpful! I designed these while I was taking microbiology class at Green River Community College. ©2018 Shirley S. Chung

Microbiology Lab Procedure Notes By ©2018 Shirley S. Chung, Green River Community College

Download these notes.

Smear

1. Clean slide
2. Label: my name, organism, date, medium

Broth Smear

1. Agitate broth unless otherwise stated
2. FLAME, 1 loopful in center of slide spread to dime-size.
3. FLAME, 2nd loopful in center of slide spread to dime-size.
4. FLAME

Solid Smear

1. FLAME, 1 loopful water (or drop)
2. FLAME, 2nd loopful water
3. FLAME 1 loopful specimen and mix well in water.
4. FLAME 2nd loopful specimen if needed and mix well in water.
5. FLAME
6. Air Dry
7. Heat Fix
8. Use clothespin.
9. Pass slide’s underside over the flame (coolest part of flame)
10. 3 passes

Simple Stain (Methylene blue, Crystal Violet, Carbol Fuchsin)

1. Smear and heat fix.
2. Flood stain

Carbol Fuchsin, 15-30 sec

Crystal Violet, 20-60 sec

Meth. Blue, 1-2 min

3. Rinse with Di Water
4. Bibulous blot, Do not wipe

Gram Stain

1. Flood with Crystal Violet
2. Wait 1 min
3. Flush with Di water
4. Flood with Gram’s Iodine mordant
5. Wait 1 min
6. Flush with Di water
7. Decolorize
8. Flush immediately
9. Counterstain Safranin
10. Wait 45 sec
11. Flush with Di water
12. Bibulous Blot

Acid-Fast Staining

1. Smear and heat fix.
2. Flood smears with carbol fuchsin
3. Place over a beaker of water on a warm hot plate
4. Steam 5 minutes (do NOT allow stain to evaporate, replenish as needed)

OR

Flood smear with carbol fuchsin WITH TERGITOL for 5-10 minutes

5. If slides are heated, allow to cool.
6. Flush with Di water
7. Decolorize with acid-alcohol
8. Flush with Di water
9. Counterstain with Meth. Blue for 2 min
10. Flush with Di water
11. Bibulous blot.

Spore Staining

1. Smear and heat fix
2. Flood with malachite green
3. Place on beaker of water sitting on hot plate
4. Steam for 2-3 minutes (do NOT allow stain to evaporate, replenish as needed)
5. Remove from hot plate
6. Cool
7. Flush with Di water (this is the decolorizing step)
8. Counterstain Safranin for 30 seconds
9. Flush with Di water
10. 10. Bibulous blot

 

Microbiology Lab Procedure Notes By ©2018 Shirley S. Chung, Green River Community College