Download VCLab 8 STANDARD PLATE COUNT

STANDARD PLATE COUNT
How do we count bacteria? There are many methods. Machines like a spectrophotometer can
measure turbidity and thereby estimate the amount of bacteria in a sample. Coulter Cell Counters
can count individual cells as they pass by a light beam. Standard Plate Count is a manual
method, also known as a population count.
Practical Applications
1. Medicine
a. Diagnosing infections: a normal urine sample is expected to have 0-300 bacterial cells
per ml. If the patient has more than 1000 cells it indicated infection. Bacteremia is the term for
bacteria in the blood that are not multiplying. Septicemia is the term for bacteria in the blood that
are multiplying. To determine the difference, we would need to do plate counts from a specimen
drawn on a later date.
2. Food Industry
a. Quality Control: All foods have some bacteria. The County Health Department has
decided what is an acceptable amount of bacterial cells per sample. A food (milk, hamburger,
etc) is labeled “Grade A” when it has less fewer than the allowed amount of bacteria. The
County does random, unannounced testing on samples.
Plate Counts are not qualitative because they do not tell you what organism is present. However,
it is quantitative because it gives you the number of organisms present.
Counting always involves math.
Try to guess how many bacterial colonies are present in a flask that is turbid from E. coli.
Write your guess down in Standard Scientific Notation.
EXPRESSING NUMBERS IN STANDARD SCIENTIFIC NOTATION
For practice: How many zero’s are there? That is the number for your exponent.
1 = 100 = 1.0 x 100
10 = 101 = 1.0 x 101
100 = 102 = 1.0 x 102
1000 = 103 = 1.0 x 103
10,000 = 104 = 1.0 x 104
Now that you’ve had practice, let’s use a random number.
Just count how many digits there are after the first one to get your exponent.
Then round off the given number to the nearest 10th (2 digits):
10634 = 1.1 x 104
Negative exponents (10-1) are fractions less than one.
Positive exponents (101) are whole numbers greater than one.
Now let’s practice using negative exponents:
Round off the third number you come to (other than zero).
For instance, if the number is 0.00342, round down the number to 0.0034
If the number is 0.00345, round up the number to 0.0035
How many digits are there to the right of the decimal point before you get to a number greater
than zero? That is your exponent number.
10-2 = 1.0 x 10-2
0.00126 = 1.3 x 10-3
0.000542 = 5.4 x 10-4
PLATE COUNT PROCEDURE
Remove a set volume of the sample with a pipette. A pipette is an instrument that measures and
delivers an accurate volume. Place the set volume sample in an empty Petri dish, add molten
nutrient agar, and mix. The E. coli have flagella and are motile, so they will spread out a bit;
however, they still tend to aggregate in clusters a bit.
Each group of cells (aggregate) forms a colony. These colonies are called Colony Forming Units
(CFU). A CFU is a cluster (aggregate) of cells which eventually leads to the formation of a
single colony. However, one single cell could also form a colony by itself. Therefore, by doing a
Plate Count, we are unable to determine how many cells are present; we only count the colonies.
Suppose we transfer 1 ml of the E. coli sample to the Petri dish, and suppose that the correct
amount of bacteria present in the solution is 2.5 x 109. That would mean there would be 2.5
billion colonies on that plate. That would be impossible to count!
COUNTABLE PLATE: 30-300 colonies
TMTC (Too many to count): 300+ colonies
TFTC (Too few to count): less than 30 colonies
To get our sample down to the number of colonies that we can count, we have to take less than 1
ml of sample. The pipettes we have can deliver 1 μl of sample, which is 1/1000 ml.
That would give us 2.5 million CFUs on the plate; still too many. Therefore, when the sample is
too concentrated, we need to do a dilution series. But how much to we dilute? 250 CFU is a
countable number.