DIRECT MICROSCOPIC AND STANDARD PLATE COUNT OF MICROORGANISMS IN MILK

DAVID YIN – 20298788

PARTNER: IKRAN ADEN

TA: BEY LING & ALEX RADEN

COURSE: BIOL 241L

LAB SECTION:  BIOL.241.104.1.LAB 

LAB TIME: TUESDAYS 7:00PM – 10:00 PM – B1 378

DATE OF EXPERIMENTS: TUESDAY, MAY 11, 2010 & TUESDAY, MAY 18, 2010

INTRODUCTION

Experimental Objective

The purpose of the experiment is to use the direct microscopic and standard

plate count methods to record evidence of bacteria growth on various milk

mediums and quantifying the results, as well as to identify how various milk

bacteria grown in the environments when the milk is raw, pasteurized,

sterilized, or post-date.

Direct Microscopic Count

The direct microscopic count is a simple examination of the various

microorganisms within the desired sample viewed under a microscope. This

involves taking a bacterial sample (in the case of the experiment, samples of

milk) and applying it onto a slide, where it can be magnified and viewed

using a standard microscope. This method is fast, simple, and does not

require substantial effort or equipment. The experimenter is able to see

individual bacteria, which makes counting individual bacteria easier, and

also shows the morphology of the microorganism. However, there needs to

be a substantial population to be counted, or the results will be inaccurate.

As well, it may be tedious and tiring for an individual to scan and count

every microorganism. Lastly, there could be dead cells, or debris and air

bubbles that look like cells; this would cause misleading results.

Standard Plate Count

The standard plate count uses agar-plates to assist in growing bacteria and

approximating growth. This involves dilutions of a sample (in the case of the

experiment, samples of milk) and pour-plating onto or mixing into agar

growth medium. After appropriate incubation, colonies will form. The

colonies are then counted – the goal is to find plates that have colony

numbers between 25-250 colonies. Under the assumption that a colony was

formed by one bacterium and using the dilution factor, one can calculate an

estimated total number of bacteria on a specific plate. This method is

advantageous because it counts only viable bacteria (those that are alive).

In addition, experimenters are able to get more accurate results for samples

with lower bacteria populations. However, only the organisms that can

thrive in the medium are grown, and so some are unaccounted for. In

addition, a colony can be formed by multiple cells, or could overlap another

colony, causing the data to be misleading. Lastly, as with the direct count

method, “non-cells” could be accidentally counted.

Microorganisms in Milk

Milk is a solution of various essential nutrients, like carbohydrates,

proteins, and fats. Thus, milk serves as an excellent growth medium for

various microorganisms. Most of these are bacteria belonging to either the

bacillus or cocci family, such as Staphylococci, Micrococci, Pseudomonas,

Flavobacterium, and Erwinia. There could also be fungi present. These

organisms are usually introduced to the milk from the producer organism.

Some specific organisms, such as Streptococcus lactis, Acinetobacter

johnsoni, and certain Lactobacilli can cause milk to become slimey or sour.

In more unsanitary conditions, disease-causing bacteria, such as

Salmonella, Mycobacterium bovis, and Staphylococcus aureus, could

contaminate the milk. Thus, pasteurization is necessary in order to make

milk safe to drink.

Pasterization Methods

Pasterization is a process that removes harmful organisms from milk, thus

lowering the overall bacterial count. This is not the same as sterilization,

which would kill all the microorganisms. The common methods are High-

Temperature Short Time (HTST) VS. Low Temperature Long Time (LTLT).

In HTST, milk is heated for at least 15 seconds at 71.6°C. In comparison,

the LTLT pasteurization heats milk for at least 30 minutes, but at 62.9°C.

After pasteurization, milk is usually chilled in refrigeration containers and

ready to be shipped and/or packaged.

MATERIALS AND METHODS

All materials and methods follow the BIOL241L manual laboratory procedures*, and there were no deviations from the norm.

EXPERIMENTAL RESULTS

A(Raw Milk)

Cells/ml

B(Fresh Milk)

Cells/ml

C(Past Date Milk)

Cells/ml

2.6 x 107 0.0 2.2 x 106

6.7 x 106 3.9 x 106 1.7 x 107

4.1 x 106 3.6 x 106 8.9 x 106

6.6 x 107 0.0 8.1 x 106

1.1 x 107 3.2 x 105 2.0 x 107

7.4 x 107 1.8 x 107 4.2 x 106

6.2 x 107 8.0 x 106 7.4 x 106

A(Raw Milk)

Cells/ml

B(Fresh Milk)

Cells/ml

C(Past Date Milk)

Cells/ml

6.7 x 106 3.9 x 106 1.7 x 107

A(Raw Milk)

CFU/ml

B(Fresh Milk)

CFU/ml

8.3 x 104 3.7 x 102

1.4 x 106 1.0 x 103

9.4 x 106 1.3 x 103

5.5 x 107 2.7 x 102

2.3 x 106 8.0 x 102

2.4 x 106 4.3 x 106

1.6 x 105 NA

6.3 x 106 5.2 x 103

2.6 x 106 3.0 x 102

7.0 x 104 4.0 x 103

1.9 x 105 NA

1.7 x 105 1.0 x 103

4.7 x 105 NA ?

7.3 x 103 NA ?

5.1 x 106 NA ?

DISCUSSION

A(Raw Milk)

CFU/ml

B(Fresh Milk)

CFU/ml

2.3 x 106 6.5 x 102

When comparing the results for the two experiments, it can be seen that,

for both experiments, the number of bacteria/colony forming units (CFU)

within raw milk are higher than numbers within fresh (pasteurized) milk.

This makes logical sense, because the pasteurization process would have

killed most of the bacteria in the milk, thus yielding a substantially lower

number. For the direct count method, the number of bacteria in fresh milk

is twice as few as the ones in raw milk. However, with the standard plate

count method, the number of bacteria in fresh milk is more than 3500 times

less than found in raw milk. In addition, the count for raw milk for both

experiments are relatively similar (both are numbers in the millions). Thus,

one can conclude that the standard plate count gives a more accurate

presentation of the bacterial population. For the direct count method,

observation errors could have accounted for more bacteria perceived than

actually existing, which would increase numbers. In addition, the counted

cells are all factored with the microscope factor, and so numbers tend to be

more dilated. It should be noted that, with the standard plate count, only

aerobic bacteria that could thrive in the medium were grown and counted.

It is possible that various organisms were unaccounted for, thus lowering

the overall count. Lastly, due to careful laboratory technique, debris and air

bubbles were relatively non-present, thus not effecting count results.

REFERENCES

APPENDIX I – RAW DATA AND CALCULATIONS