Part I. SERIAL DILUTION AND VIABLE COLONY COUNT OF PASTEURIZED MILK
PREPARATION AND MATERIALSSmall carton of fresh pasteurized milk per class (if a source of raw milk is available, let some teams use it and compare results with those teams using pasteurized milk),
Three 9-ml water blanks per team
Three agar plates per team
Four sterile disposable 1ml pipettes per team
Spreading rods
Colony counter available for next (evaluation) lab
STUDENT INSTRUCTIONS
Working in teams of two or four, as indicated
by the instructor, follow the flow chart sketched on the board.
-Use 1 ml pipettes for all transfers, change pipettes at asterisks. In
all, you will use and discard 4 pipettes.
-Gently spread ˝ ml (from pipette) across the agar surface as evenly as
possible with the bent glass rod or steel spreader.
[Students often confuse the numbers on the pipette. The entire pipette filled to the 0 contains only one ml. To inoculate with ˝ ml, let the pipette empty down to the .5 ml mark (half the contents). You may want to fill the pipette only halfway full in the first place].
The lighted counting grid may help.
Multiply the number of colonies by the dilution factor of that plate.
Ex: if the 1:200 plate shows 148 living colonies:
148 x 200=29,600 bacteria in ml of the original sample
Gram stain a sample colony, observe and record results by sketching.
Milk is normally sterile as it forms in the udder of the cow. Contamination occurs as the milk is passed through the ducts of the udder. Lactobacillus and Streptococcus species become milk contaminants at this point. These are normal in raw or pasteurized milk and rarely would be involved in pathegenesis. They do, however, contribute to souring of milk. Cool temperatures prolong the palatability of such milk. Additional contamination can occur from dust, manure, polluted water, poor processing techniques such as unclean mechanical equipment and unsanitary handling of the dairy products by personnel. Common contaminants are Pseudomonas, Clostridium, Flavobacterium, Bacillus, and Micrococcus. If the cow is infected with a pathogenic organism such as Mycobacterium sp., the milk provides a means of transmission to humans.
Pasteurization involves heating the milk to temperatures, which would eliminate fastidious pathogens, but not alter the food value and palatability of the milk. Such heating does not sterilize the milk, as the Lactobacillus will survive the temperature. However, the bacterial count is greatly reduced and the pathogens are killed.
For Grade A Government (USDA) inspected milk,
pasteurized milk should not contain over 20,000 bacteria/ml. Quality raw
milk for distribution or subsequent pasteurization may not contain over 200,000
bacteria/ml. Coliform bacteria may not exceed 10/ml.
PREPARATION AND MATERIALS
Students bring various environmental water
samples for testing
(spring water, creek water, pond water, and well water make interesting
comparisons)
Provide one EMB plate and two phenol red lactose broth tubes for each pair of students.
STUDENT INSTRUCTIONS
With the environmental water sample provided, streak for isolation on an EMB plate. This type of agar contains the dyes eosin and methylene blue. They inhibit Gram-positive organisms. Such a medium is selective for Gram-negative species.
Lactose-fermenting organisms such as E. coli produce a black precipitate on EMB. Their colonies will be either black or possess dark centers with transparent, colorless peripheries. Non-lactose fermenters such as Proteus sp., Salmonella sp., or Shigella sp. appear pink or uncolored. Thus, the medium is considered differential with respect to lactose fermentation.
Following incubation for 48 hours, pick a non-lactose fermenter and transfer for pure culture to lactose broth. Do the same with a lactose-positive colony. Incubate these tubes for 48 hours and compare results. Also, Gram stain from the EMB colonies to confirm that the medium selects for Gram-negative organisms.
Gram positive contamination of water can be from natural contamination through soil. Gram positives found in water are not generally associated with fecal contamination or pathogenesis. Specific identification of these organisms is not necessary. However, large numbers of gram negative organisms in a water supply do merit further testing since many of the enteric bacteria are gram negative and the group does contain significant pathogens. If Gram negatives are found in a water supply, the next question to be answered is whether they are lactose positive or negative. If they are strongly lactose positive with gas production, this result can be presumed to indicate fecal contamination. Escherichia coli (Gram-, Lactose+, Indole+) is the indicator organism for fecal contamination, since its “habitat” is the intestinal tract of man and animals. Lactose negative organisms can include some enteric normal flora such as Proteus, but also some true pathogens such as Shigella and Salmonella.
Look up a description of SIM medium in the manuals available in the lab room in the library.
Using SIM medium, Simmons Citrate, Dextrose Broth, and Urea Agar, design an elimination plan, which would differentiate between Escherichia, Enterobacter, Proteus, Salmonella, and Pseudomonas.
I. LACTOSE POSITVE
A. INDOLE POSITIVE: Escherichia
B. INDOLE NEGATIVE: Enterobacter
II. LACTOSE NEGATIVE
A.. GLUCOSE POSITVE
1. MOTILE
a.
UREASE POSITIVE: Proteus
b.
UREASE NEGATIVE: Salmonella
2. NON-MOTILE and
SULFIDE NEGATIVE: Shigella
B. GLUCOSE NEGATIVE: Pseudomonas (can
fluoresce under UV light)