FST 3201- 3 (BASIC FOOD MICROBIOLOGY)

EXPERIMENT TITLE :
MICROBIAL ENUMERATION

EXPERIMENT DATE:
4 MAY 2023

LECTURER’S NAME:
PROF. MADYA. DR. FARINAZLEEN BINTI MOHAMAD GHAZALI
PROF. MADYA. DR YAYA RUKAYADI

MEMBERS OF SUBGROUP 6

NAME MATRIX NUMBER

NOR SYUHADA BINTI BAHARUDIN 210374

NURUL AISYAH BINTI ABDUL RAHMAN 209903

NOR SYAZANA BINTI ZAINUDDIN 210216

FARAH SOFEA BINTI SUHAIMEE 209799

MARKING BOX:
ABSTRACT

This experiment demonstrates several ways of enumerating microbial cells which are

plate count method as a direct variable count which is commonly used for measuring bacterial

populations, most probable number method, direct microscopic count method and turbidity

method which can be done without spoiling the sample in order to determine their numbers.

Students will be able to choose and perform a suitable method for enumeration when given a

sample as it is essential to determine the number of microorganisms in a given sample.

Besides, this experiment requires students to be proficient in collecting, tabulating, analyzing,

interpreting and evaluating the experiment data. Lastly, The techniques used in this experiment

are useful in the food and beverage industries, where counting the number of bacteria in a

specific food or beverage sample is necessary to determine whether the food or beverage is

safe to ingest and not contaminated.

OBJECTIVE

The aims of this experiment are to implement and conduct various techniques of

enumerating microbial cells. Aside from that, the ability to choose an appropriate enumeration

technique when provided a sample. Finally, to collect, tabulate, and examine, assess and

evaluate the given experimental data

INTRODUCTION

Enumeration is the process of counting the amount of bacteria in a specific sample. In this

laboratory session, there will be three techniques used in microbial enumeration which are plate

count, most probable number and direct microscopic count / total cell count (using

haemocytometer).

Firstly, plate count is a count of viable or live cells and it is done by diluting the liquid sample

and plating the dilutions onto a suitable growth medium by spread plating / dilution plating.

There are two types of plate count which are total plate count to count viable bacteria and yeast

and mould count to count viable fungi.

Next, most probable number is a sample diluted in a series of tubes containing broth. The

higher the number of cells, the higher dilutions it takes to dilute the cells out entirely. For the

results of such dilutions, they can be compared to statistical tables where a cell count can be

estimated. So, to increase the statistical accuracy of the most probable number, there will be

more than one tube that can be inoculated from each dilution.

Lastly, direct microscopic count where the most simplest means accurately to determine the

number of cells by using haemocytometer. Looking through the microscope, choose which

square that wants to be counted to the sample. However, there will be error in using

haemocytometer counts like statistical errors, improper filling of chambers and non-uniform

suspensions.
MATERIAL AND METHODS

A)PLATE COUNTS

MATERIALS
Alcohol, Colony counter, Escherichia coli broth culture (12 hours old), Glass spreader, Nutrient

agar plates, Pipette ( 1 mL, 0.1 mL ) and pipette tips, Universal bottles containing 9 mL diluent (

either distilled water or peptone water).

METHODS
−1 −2 −3 −4 −5
Firstly, Universal bottles containing 9 mL diluent with 10 , 10 , 10 , 10 , 10 and

−6
10 were labelled. Secondly, the E.coli culture was gently shaken to ensure even suspension,

−1
1 mL of the culture was pipetted into the 10 tube, and the tube was shaken by using a vortex

−1 −2
mixer. Thirdly, 1 mL of the culture solution was transferred from 10 tube into the 10 tube by

using fresh pipette tips and was shaken well by using a vortex mix. Then, step 3 was repeated

−3 −6
for tube 10 until 10 . This step we called a serial dilution. Next, the nutrient agar plates with

the corresponding dilution factors were labelled, in duplicates. 0.1 mL of each dilution factor

were transferred onto the centre of the nutrient agar surface. The glass spreader were sterilised

by dipping it in alcohol and flamed in the Bunsen flame. After that, 0.1 mL aliquot was spreaded

evenly on the surface of nutrient agar by using the sterilised glass spreader. Then, nutrient agar

plates were then incubated at 37°C for 24 to 48 hours. Lastly, the number of colonies were

counted after incubating using a colony counter with a selected plate with acceptable range of

colonie and the CFU were calculated.

B) MOST PROBABLE NUMBER

MATERIALS
Pipette (1 mL, 0.1 mL) and pipette tips, Serial dilution of Escherichia coli culture prepared in

section (A), Test tube containing 10 mL Nutrient broth (NB).

METHODS

−0
Firstly, Three nutrient broth tubes with dilution factor were labelled 10 ,

−1 −2 −3 −4 −5 −0
10 , 10 , 10 , 10 and 10 . In total, there will be 18 tubes. Secondly, 1 mL from 10

−0
E.coli culture was transferred into 10 nutrient broth tubes in triplicates. Next, step 2 then was

−1 −2 −3 −4 −5
repeated for 10 , 10 , 10 , 10 and 10 . Then, nutrient broth tubes were then incubated

at 37°C for 24 to 48 hours. After that, take note of the nutrient broth tubes after incubation.

Cloudy tubes are positive, while clear tubes are negative. Tabulated the results, and the dilution

to extinction three digit code, were obtained and read it against a 3-tube MPN table.The number

of viable cells/mL of the original culture were then calculated.

C) DIRECT MICROSCOPIC COUNT

MATERIALS
Coverslip, Dropper, Haemocytometer slide, Microscope, Yeast culture

METHODS

Firstly, a drop of yeast culture was placed onto the haemocytometer, and the coverslip

was placed on it. Secondly, the depth of the haemocytometer was taken and it was useful for

final calculation. Then, All the cells were counted within the counting area consisting of 25

medium squares. Lastly, the number of yeast cells/mL then were calculated.
RESULTS

(a) Total Plate Count

Table 2.0: The Total Plate Count of Escherichia coli at different dilution factors on
Nutrient agar plates following incubation at 37॰C for 24 hours. Aliquot = 0.1 mL

Calculation = (No. of colonies x dilution factor) ÷ aliquot

Dilution Replicate 1 Replicate 2 Calculation CFU/mL

Pure culture - - - TNTC

−1 - - - TNTC
10

−2 - - - TNTC
10

−3 - - - TNTC
10

−4 262 262 5 2.53 x 10

8
10 (253 x 10 ) ÷ 0.1

−5 132 176 6 2.53 x 10

9
10 (154 x 10 ) ÷ 0.1

TNTC : Too numerous to count

TFTC : Too few to count
 (b) Most probable number
Table 2.0 : The Most Probable Number of Escherichia coli at different dilution factors in
MacConkey broth following incubation at 37॰C for 24 hours

Dilution Tube 1 Tube 2 Tube 3 Calculation MPN/mL

Pure culture G G G 3 -

−1 G G G 3 -
10

−2 G G G 3 -
10

−3 G G G 3 -
10

−4 G NG G 2 1.1 x 10
2
10

−5 NG NG G 1 -
10

Indicator:
G: growth, NG: No growth

(C) Direct microscopic count

Selected square : small

Area of selected square : 0.0025 (mm)^2
Depth of haemocytometer : 0.1 mm
Volume of square : 0.00025 mm
0.00025 mm3 = 0.00025 µL

Square replicate 1 : 3 cell

Square replicate 2 : 4 cells
Square replicate 3 : 5 cells
Average 4,000 cells in 1 µL : 4 cells in 0.00025 µL

6
Cell count: : 4,000,000 = 4 × 10 cell/mL
DISCUSSION

In this experiment, we learn how to do plate count, which is a count of viable or live cells. To

make the counting of the colony effective, the dilution of the sample must be grown in between

30 and 300 colonies. Less than 30 colonies makes the sample unsound and greater than 300

colonies probably resulted from the overlapping colonies and might cause imprecision of count.

The advantages of the viable cell count are, it can count viable cells in any type of samples and

we can see the cell morphology. But, it requires a long time to observe the cell growth because

of the incubation period. Besides, the colonies can be hard to count because of the clumping or

chains of cells. Meanwhile, the second method, total cell count estimates all cells including both

living and dead microbial cells in a sample. This method is easy, inexpensive, and does not

need incubation time. But the disadvantages are, it cannot differentiate dead and living cells,

difficult to identify small cells, and hard to achieve precision.

The common antibacterial and antifungal substances added to the growth media is liquid

inoculum. DRBC enumeration medium contains dichloran, rose Bengal, and chloramphenicol.

Dichloran and rose bengal significantly reduce the size and height of mould colonies, which

helps to accurately count colonies and prevents the overgrowth of the species. Most bacterial

growth is suppressed by the addition of chloramphenicol, allowing moulds and yeasts to grow.

In the experiment, 0.1 mL was plated with the spread-plate technique so that the inoculum from

the larger volume would not be soaked into the agar and causes the colonies to stick or grow at

the edge of the petri dish. Meanwhile, the 1.0 mL was plated with the pour-plate technique so

that the colonies can grow well on the agar surface and provide convenient means to count the

cell’s sample. Plates with fewer than 30 colonies are designated too few to count (TFTC), while

samples with more than 300 colonies are considered too numerous to count(TNTC). If TFTC or
TNTC occurs, the sample is considered invalid and usually discarded. If the MPN method yields

all positive results, a confirmatory test can be performed. It verifies the presence of coliform by

examining the positive test tubes. The MPN gas method does not prove the presence of

coliform in the water sample. Yeasts and Clostridium species might be present in the water, and

they can ferment lactose to produce gas and acid. Therefore, the detection of coliform in water

must therefore be confirmed.

CONCLUSION

To conclude, at the end of this experiment, we finally can learn that there are different ways to

enumerate microbial cells which are plate count, most probable number and direct microscopic

count and when to use them. Next, we learn how to dilute the colonies in plate count method

and how to calculate MPN.

REFERENCES

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