COLLEGE OF ARTS & SCIENCES

Natural Sciences Department

STANDARD PLATE COUNT

by: Carl Errol S. Seran

Background Information

The enumeration of the number of bacteria in a specimen is essential to ensure quality control
in industrial and applied microbiology and food safety. Several methods can be used to
determine bacterial concentrations. These include direct counts, plate counts, ltration, and
turbidimetric measurements. The plate count is one of the most accurate means of
enumeration of viable microbes because you get a visual indicator for every cell in the
specimen. The technique stems from Robert Koch's insight gained from viewing colonies
growing on the surface of a spoiling slice of potato.

The pour plate technique can be used to determine the number of microbes/ml or microbes/
gram in a specimen. It has the advantage of not requiring previously prepared plates, and is
often used to assay bacterial contamination of foodstu s. Each colony represents a "colony
forming unit" (CFU). For optimum accuracy of a count, the preferred range for total CFU/plate
is between 25 to 250 colonies/plate.

Materials and Methods

Water Bath, thermostatically controlled to 45 ± 10C

Colony Counter with grid plate
Incubator set at 370C
Vortex mixer

Escherichia coli suspension

PER GROUP
Automatic Pipette (1 ml)
Sterile pipette tips
1 Tally Counter
1 alcohol lamp
2 inoculating loop

6 Sterile Petri Dishes

6 Sterile Culture tubes with stopper
100 ml sterile distilled water (Dilution blanks)
100 ml melted EMB, thermostatically controlled at 45 ± 10C
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 COLLEGE OF ARTS & SCIENCES
Natural Sciences Department

STANDARD PLATE COUNT (Pour Plate Method)

1st LAB HOUR

Serial Dilution and Plating

1. Liquefy a bottle of 100 ml EMB.

2. While EMB is being heated, label the 5 sterile culture tubes with the respective dilutions
(10-1, 10-2, 10-3, 10-4, 10-5). Do the same with the 5 sterile empty plates.
3. Transfer 9 ml of sterile distilled water to each of the labelled sterile tubes.
4. Shake the bacterial suspension and transfer 1ml to the rst tube (10-1), using a sterile 1 ml
pipette. After using the pipette, discard properly.
5. Shake the blank using a vortex mixer. Forceful shaking not only brings about good
distribution, but it also breaks up clumps.
6. With a di erent 1 ml pipette, transfer 1 ml of Blank 1 to Blank 2 making 10-2 dilution.
Repeat the procedures 4 to 6 with the remaining tubes (Blank 3, 4, 5) to make the desired
dilutions (10-3, 10-4, 10-5)
7. With another sterile pipette, transfer 1 ml from Blank 1 to the 10-1 plate. Repeat the
procedure with their respective dilution plates (Blank 2 for 10-2 plate, and so on…)
8. After the bottle of nutrient agar has melted, cool it down in a water bath, thermostatically
controlled at 45 ± 10C for at least 10 minutes.
9. Pour approximately 20 ml of nutrient agar into each of the ve plates with dilutions. Rotate
the plate gently to get adequate mixing of the medium and organisms. This step is
critical! Too little action will result in poor dispersion and too much action may slop the
inoculated medium over the edge.
10. After the medium has cooled completely, invert the plate and incubate at 35 - 370C for 24 -
48 hours.

2nd LAB HOUR

Counting and Calculations

1. After incubation, lay out the plates on the table in order of dilution.
2. Place each plate on the colony counter in their respective dilution. Start counting at the top
of the plate, using the grid lines to prevent counting the same colony twice. Use a tally
counter. Count every colony, regardless of how small or insigni cant. Record your counts.
3. Select the plates that have between 25 - 250 colony count. Plates with less than 25 or
more than 250 are statistically unreliable. Report TNTC for plates with more than 250
colonies, and TFTC for plates with less than 25 respectively.
4. Calculate the number of bacteria per ml of undiluted culture. Multiply the number of
colonies counted by the dilution factor (the reciprocal of the dilution).

Example: If you counted 220 colonies in the plate that received 1 ml of the 1: 1000 dilution (10-4): 220 x
1000 (or 2.2 x 105) bacteria per ml.

Use only two signi cant gures. If the number of bacteria per ml was calculated to 227 000, it should be
recorded as 230 000 or 2.3 x 105
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