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
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.
Complete the Bacteria Growth Characteristics Laboratory Investigation.
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.
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