Sometimes students are required to write a proposal for a required research project. Other teachers may not require a written proposal, the student may want to write a proposal so he has a carefully planned proposal as a guideline.
Once the project is begun and the student learns more, he will likely find ways to modify the project. Hopefully, the teacher will permit modifications of the work plan.
Most students are aware that raw milk has some manure, dust from hay, bacteria from the milk bucket, as well as the possibility of bacteria from infections such as undulant fever, brucellosis, and mastitis. This proposal has been written from the viewpoint of a student who thinks it would be interesting to see what kinds of bacteria are found in a sample of fresh raw milk which was milked from the cow as in the olden days. While anerobic bacteria may be present, the student does plan to isolate any anaerobes.
Safety: since the farm family has been drinking the milk, it would seem that presence of pathogenic bcteria is unlikely, but the student should consider that possibility and list the diseases which might be present. Never ingest, inhale, or rub eyes with the organisms which you isolate.
Raw milk obtained from a cow by hand milking in a typical cow stable may contain bacteria from the cow's udder and hair, flies that fall into the bucket, bacteria that are growing in the poorly washed seams of the bucket, dust from manure and hay, dirt from the milker's hands, cow's tail dipping into the bucket, etc. Lactic acid bacteria, Pseudomonas, and other bacteria are common in such milk. If the udder has mastisis specific bacteria may be present.
I do not have the time nor the selective media to grow all the kinds of bacteria which might be present in the milk. I will make no attempt to isolate bacteria which can't grow in the presence of air. I will use tryptone-glucose-yeast extract agar for the initial isolation. This medium is often called plate count agar because large numbers of genera of bacteria are able to grow on it. Some bacteria present in the sample may not be able to grow on TGY agar.
I plan to study some of the bacteria hoping to determine the genera, or general group to which they belong, but I will be limited by time and available assay media. My main goal in the characterization of the pure cultures I isolate is to see the results of common assays such as gram stain, fermentation of various sugars, and other traits commonly used in classification of bacteria from milk. .
To isolate under aerobic conditions the predominant bacteria found in raw cow's milk and study the traits of ten of the bacteria using the limited number of diagnostic media I have available.
Isolation of bacteria: The raw milk will be streaked on TGY Agar (Tryptone-Glucose-Yeast extract) and the plates incubated under aerobic conditions at room temperature (20 to 30C). Ten colonies having differing appearances will be selected for further characterization.
TGY Agar, also called Plate Count Agar, is commonly used for counting bacteria in food and environmental samples because it supports the growth of many common bacteria. Only the predominant bcteria will be found by this method. Selective methods such as adding bile to the medium could be used in later studies if special groups of bacteria were to be isolated. Since bile is found in the intestine, using bile in the medium inhibits the growth of many common bacteria but has little effect on he growth of common gut organisms.
Characterization of Isolated Strains: I plan to characterize each of the ten isolates by as many of these tests as time, equipment, and materials permit.
Oxygen Requirement: aerobic, microaerophilic, facultative anaerobic.
Temperature Optimum or limit: 4C (in refrigerator), 20 - 30 C, 37 C if incubator available
Cell shape: coccus, bacillus (rod), spiral.
Cell grouping: single, chains, packets, filaments, sheets, etc.
Ability to grow on mineral medium (organotrophic, vs lithotrophic)
Gram stain (+ or -); if gram stain is available
Spore production; assay pm SPA at room temperature.
Metabolism: oxidative vs fermentative
catalase. Many bacteria decompose produce bubbles of oxygen from hydrogen peroxide
Assignment to Genus or Broad Group: Based on the assay results I hope to tentatively assign each isolate to one of the broad groups given in Bergey's Manual of Determinative Bacteriology (1974). I may continue this project as a hobby and attempt to key one or more of the isolates down to Genus but due to lack of diagnostic media and time. I do not expect to have sufficient information about any strain to assign it to Genus during this semester.
1. Because the bacteria are being isolated from milk on organic medium after brief incubation under aerobic conditions, I do not expect any isolate to be phototrophic, gliding, lithotropic, anaerobic, etc.
2. Isolates from milk might fall into one or more of these Bergey groups:
3. There is less chance they will be : aerobic spore formers.
This file was completed 1999 Jan 26 at 11:00 a.m.
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