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Medical microbiology

BACTERIA GROWTH CHARACTERISTICS
OBJECTIVE/RATIONALE
Recognizing colony growth characteristics and biochemical reactions to various media is important in identifying bacteria. The student will plate known bacteria and observe for colony characteristics and biochemical reactions. TEKS 121.14 (c) 1A, 1B, 4B, 4C, 4D National Science Education Standards A9-12;C9-12 National Health Care Skills Standards .01, .04, .05, .06, .07, .08 National Curriculum Standards for School Mathematics S1; S3 KEY POINTS
Introduction a. Bacteria have different characteristic morphology of their colonies. These colonies will take on additional characteristics that aid in differentiation depending on the media/agar used for growth. Colony characteristics: a. The Colony Surface 1. Smooth 2. Rough 3. Wrinkled 4. Glistening a. Soluble: Does the color diffuse into the media/agar? b. Insoluble: Does the color stay within the colony? c. Color: white-white, white-gray, light purple, yellow-brown, etc. 1. Take an inoculating loop and poke at the colony. Describe it by one of the following: a. Mucoid: resembling mucous b. Dry: crusty, brittle c. Soft: butter-like consistency 1. Translucent: See-through 2. Opaque: No light shines through 3. Iridescent: Rainbow colors in reflected light 4. Dull: No shine to the colony 5. Shiny: Reflects light e. Shape of the colony (See handout for pictures) 1. Circular 2. Irregular 3. Spindle 4. Filamentous 5. Spreading f. Height of colony (See handout for pictures) 1. Raised 2. Flat 3. Convex 4. Heaped 5. Sunken Media and Biochemical Reactions A. MSA-Mannitol Salt Agar: a selective medium for the isolation of Staphylococci species. It contains a 7.5% NaCl that inhibits the growth of most gram-negative and gram-positive microorganisms. MSA contains beef extract as a nutrient and phenol red indicator. When Staphylococcus aureus is grown, it ferments the sugar mannitol, producing yellow colonies. Those Staphylococcus species that do not ferment mannitol, remain as small red colonies and the plate remains pink to red. B. MAC-MacConkey: a selective, differentiating medium for the isolation of gram-negative bacilli and differentiation of lactose fermenters from nonlactose fermenters. It contains bile salt and crystal violet that inhibit the growth of gram-positive bacteria. If the microorganism can ferment lactose, the colonies appear pink to red in color and if it cannot ferment lactose, the colonies appear clear. The medium also contains lactose and neutral red indicator. C. BLD-Sheep blood agar: an enriched, non-selective medium. It is trypticase soy agar with 5% sheep red blood cells added to it. Gram-positive and gram-negative bacteria can grow on this medium. Hemolytic patterns of certain bacteria may be seen on the plate: 1. Alpha hemolysis: incomplete hemolysis; greening of the medium 2. Beta hemolysis: total clearing of the medium 3. Gamma hemolysis: nonhemolytic; no change in the color of the D. EMB-Eosin methylene blue agar: a selective, differential medium for the isolation of gram-negative bacilli and differentiation of lactose fermenters from nonlactose fermenters. The medium contains lactose, eosin dye, and methylene blue dye. The dyes are toxic to gram-positive bacteria. When bacteria ferment the lactose, the pH decreases in the medium and the medium changes to a purple color. Gram-negative bacteria that ferment lactose appear with a green metallic sheen. Those that cannot ferment lactose appear clear on the medium. E. CNA-Colistin-nalidixic acid agar: for isolation of gram-positive bacteria. The medium contains sheep blood agar and the antibiotics colistin and naladixic acid added. Colistin disrupts the cell membrane of gram-negative microorganisms and the nalidixic acid blocks DNA replication in the gram-negative bacteria. F. PEA-Phenylethyl alcohol agar: selective medium that inhibits the swarming of Proteus species. Gram-positive facultative anaerobes such as Staphylococcus, Streptococcus, and Corynebacterium grow on this medium. G. TSA-Trypticase soy agar: a general purpose medium for isolation of fastidious microorganisms. The medium contains nutrients for both gram-negative and gram-positive bacteria. H. CHOC-Chocolate agar: an enriched non-selective medium to isolate fastidious microorganisms. Neisseria species and Haemophilus species grow well on this medium. This medium is heated to destroy red blood cells and release NAD to aid in growth of bacteria. It also has IsoVitaleX, which contains dextrose, cysteine, vitamin B12, thiamine, and ferric nitrate. I. TSB-Trypticase Soy Broth: a general purpose medium for all types of microorganisms. This medium is in a liquid form that is great to use for growth of stock cultures. J. UREA BROTH: for identification of urease producers, such as Proteus, Morganella, Klebsiella pneumoniae, and some Enterobacter species. The enzyme, urease, splits urea into alkaline end products. This results in an increased pH of the medium and a color change in the indicator to pink-red. K. GN BROTH: an enrichment broth used to inhibit gram-positive microorganisms. It contains bile salt that is toxic to gram-positive bacteria and is inhibitory to normal flora coliforms of the digestive system. GLOSSARY OF TERMS 1. Differential media: primary isolation media that is selective for a specific 2. Enrichment media or broth: enhances the growth of groups of bacteria. 3. Fastidious: bacteria that have complex nutritional requirements. 4. Hemolysis: destruction of red blood cells. 5. Coliforms: gram-negative bacilli that ferment lactose, i.e. Escherichia coli. Important Point to Remember a. Examine the colonies with light sources above and below the plate. b. Color description should be extremely specific. c. Examine well isolated colonies. d. Check for changes in appearance of the media/agar surrounding the colony. e. Note any unusual characteristics, i. e. a fried egg appearance of the colony. f. Enrichment media may cause some colonies to grow larger than others and some dyes may cause the colonies to be brightly colored. ACTIVITIES
Complete the Bacteria Growth Characteristics Laboratory Investigation. MATERIALS/RESOURCES
Bacterial Morphology Handout Bacteria to be used:
Inoculating loop
Bunson burner
Gloves
Goggles
Incubator 37C
Lab coats
Disinfecting Solution
Paper towels
ASSESSMENT
ACCOMMODATIONS
For reinforcement, the student will design a chart of colony characteristics and biochemical reactions. For enrichment, the student will identify an unknown bacteria using colony characteristics and biochemical reactions. REFLECTIONS
BACTERIALS MORPHOLOGY

COLONY SHAPES:
Circular

Irregular

Filamentous

Spreading
COLONY HEIGHT:

Heaped Sunken

BACTERIA GROWTH CHARACTERISTICS
LABORATORY INVESTIGATION
PURPOSE:
In this laboratory investigation, the student will plate known bacteria and observe for colony
characteristics and biochemical reactions.
BACKGROUND INFORMATION:

MATERIALS:
Bacteria listed below
Media Listed below
Inoculating loop
Bunson burner
Gloves
Goggles
Incubator 37C
Lab coats
Disinfecting Solution
Paper towels
PROCEDURE:
Plate the following bacteria on the list of media printed below, using the plating
technique you have learned. Be sure to use standard precautions and treat every
specimen as a potential pathogen.
Bacillus subtilis
Escherichia coli
Proteus mirabilis
Pseudomonas aeruginosa
Enterococcus faecalis
Micrococcus luteus
Staphylococcus aureus
Staphylococcus epidermidis
Bacillus cereus
Serratia marscens


Media to be used:
MSA
MAC
BLD
EMB
CNA
PEA
TSA
CHOC
TSB
UREA BROTH
GN BROTH
DATA: Record the growth, colony characteristics, and biochemical reactions (if
applicable) for each of the following bacteria:
Bacillus subtilis:
MSA:

GN BROTH:
Escherichia coli
MSA:

Proteus mirabilis
MSA:

GN BROTH:

Pseudomonas aeruginosa
MSA:

GN BROTH:

Enterococcus faecalis
MSA:

Micrococcus luteus
MSA:

GN BROTH:

Staphylococcus aureus
MSA:

GN BROTH:

Staphylococcus epidermidis
MSA:

Bacillus cereus
MSA:

GN BROTH:

Serratia marscens
MSA:

GN BROTH:

CONCLUSION:
Prepare a cross reference list of each bacteria that grew on each plate.


Source: http://medmicro.pbworks.com/f/bacteria_growth_characteristics.pdf

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