CHAPTER 5

METHODS OF ENUMERATING MICROORGANISMS OF FOODS

Methods of enumerating microorganisms of foods are mainly bacteriological, since foods are most often spoiled because of bacterial growth.

However, yeast, molds and virus sometimes cause food spoiled.

There are several ways to enumerate microorganisms:

a) Total plate count

b) Coliform test

c) Testing for mesophilic bacteria

d) Testing for staphylococci

e) Testing for pathogenic bacteria: eg - Salmonella, Shigella

A variety of culture-based techniques can be used to determine the number of microbes in a sample.

A major assumption of such methods is that, under suitable condition, an individual viable microbial cell will be able to multiply and grow to give a visible change in the growth medium eg: a colony on agar based medium or turbidity (cloudiness) in a liquid medium.

TOTAL PLATE COUNT

Many culture methods make use of a solidified medium within a petri plate.

The three most important procedures involving in plate counts are:

1) Streak dilution plate

2) Spread plate

3) Pour plate

a) Streak dilution plate

Streaking a plate for single colonies is one of the most important basic skills in microbiology.

A sterile inoculation loop is used to streak the organism over the surface of the medium (Figure 13.1)

The aim is to achieve single colonies at some point on the plate.

Single colonies containing cells of a single species and derived from a single parental cell.

Procedures:

Keep the lid of the petri plate as close to the base as possible to reduce aerial contamination.

Allow the loop to glide over the surface of the medium. Hold the handle at the balance point, sweeping movements, as the agar surface is easily damaged and torn.

Do not breath directly onto the exposed agar surface and replace the lid as soon as possible.

b) Spread plate

This method is used with cells in suspension, either in a liquid growth medium or in appropriate sterile diluents.

It is one method of quantifying the number of viable cells (or colony forming units) in a sample after appropriate dilution.

An L-shaped glass spreader is sterilized by the end of the spreader in a beaker containing a small amount of 70% v/v alcohol, allowing the excess to drain from the spreader and then igniting the remainder in a Bunsen flame.

After cooling, the spreader is used to distribute a known volume of cell suspension across the plate (Figure 13.2)

3) Pour plate

This procedure also uses ceils in suspension, but required molten agar, usually in screw-capped bottles containing sufficient medium to prepare a single Petri plate (i.e. 15-20 ml), maintained in a water bath at 45°C - 50°C.

A known volume of cell suspension is mixed with this molten agar, distributing the cells throughout the medium.

Then is then poured without delay into an empty sterile Petri plate and incubated, giving widely spaced colonies (Figure 13.3)

Colonies are formed within the medium, they are far smaller than those of the surface streak method, allowing higher cell numbers to be counted.

Disadvantages:

1) Typical colony morphology seen in surface grown cultures will not be observed for those colonies that develop within the agar medium.

2) Some of the suspension will be left behind in the screw-capped bottle

Count the accurate result which contain 30 - 300 colonies

↓

Count the mean

↓Calculate the colony count per ml of that particular dilution by dividing by the volume (in ml) of liquid transferred to each plate

(v)

Example: Count per ml (unit in CFU) = C/V x M

C= Mean of colony eg: 5.5 colonies V= Volume of transferred to plate eg: 0.05ml M= Multiplication factor eg: for a dilution of 10'7, the multiplication factor would be 107

or in other words: mean colony count of 5.5 colonies per plate for volume of 0.05ml at a dilution of 10'7, the count would be:

5.5 / 0.05 x 10^ 1.1 xlO^FUmI-1

COLIFORM TEST

What is coliform bacteria ?

Coliform bactria is a gram negative nonspore forming bacilli usually found in the human and animal intestine. They ferment lactose to acid and gas

Coliform test been carried out to test the presence of enteropathogenic bacteria such as Salmonella, Shigella and others

The test normally using coliform bacteria as indicator Why coliform bacteria?

Because they are able to survive for extensive periods of time in the environment, relatively easy to cultivate in the laboratory and numerous. E.coli is the most important indicator organism within the group

The ideal indicator organism should:

a) Be present whenever the pathogens concerned are

present

b) Be present only when there is a real danger of pathogen

being present

c) Occur in grater numbers than the pathogen to provide a

safety margin

d) Survive in the environment as long as the potential

pathogens

e) Be easy to detect with a high degree of realibility

Test for conforms involves 3 stages:

a) Presumptive tests

b) Confirmation tests

c) Completed tests

a) Presumptive test

Example in solid sample like food

Use plating method

Food is added to petri dishes (in duplicated)

Then molten violet red bile agar is added, mix, then allowed to harden

The plate then incubates at 35°C for 18-24 hours.

Positive result: dark red colonies with a surrounding zone of precipitated bile at least 0.5mm in diameter

Example for drinking water:

Most Protable Number (MPN) method

In this method, samples were inoculated in 10ml (101), 1ml(100) and 0.1ml (10-1) into lactose both tubes.

The tubes are incubated-and coliform organisms are identified by their production of gas from lactose.

Referring to an MPN table, a statistical range of the number of coliform bacteria is determined by observing how many broth tube showed gas.

However presumptive test for coliforms does not constitute absolute identification of these organisms. All positive result should be confirmed by further testing which is confirmation test.

B) Confirmation test

This test being carried out because gas formation in lactose broth at 37°C is characteristic not only of fecal Salmonella, Shigella and E.coli strains but also of non fecal coliform like Enterobacter aerogenes and some Klebsiella species.

As a result, in this second stage test (confirmation), the presence of enteric bacteria is confirmed by reculture the positive result:

Positive result from plating method:

Those colonies from violet red bile agar are transfer to a separate tube of brilliant green lactose bile broth and then incubate for 48 hours under 35° C before examined for the gas presence.

d) Completed test

Positive tube from Brilliant Green Lactose bile broth cultures are streaked and stabbed on slant of nutrient agar

After incubation for 18-24 hours at 35 °C, the slant is examined for the growth on the surface and in the stabbed portion of the slant

Gram stain smear was made from agar slant:

Positive result showed: Gram negative, non-sporing rods

TEST FOR MESOPHILLIC BACTERIA

MesophHIic bacteria is a bacteria that can grow at the optimum temperature (between 20 °C- 50°C).

However many mesophiles have an optimal temperature of about 37° C, corresponds to human body temperature.

Many of the normal resident microorganism of the human body, such as Escherichia coli are mesophiles.

TESTING METHOD FOR MESOPHILIC BACTERIA

Aliquots from serially diluted sample/suspensions are either pour or surface plated using non selective media such as Plate Count Agar (PCA), tryptic soy agar, nutrients agar and others.

However, PCA is recommended for colony forming units

(CFU) determination.

The temperature and time of plate incubation required for the colonies to develop differ with the microbial groups being enumerated.

For mesophilic bacteria, normally incubation temperature is 37ºC for 24-48 hours.

TESTING FOR STAPHYLOCOCCI

Why Staphylococci?

Because certain strains of Staphylococc\ like S.aeureus can grow on food and produce heat-stable enteroxins, which cause food poisoning.

Characteristics of Staphylococci

1) Gram positive, cocci arranged in clusters.

2) Biochemical test: Oxidase (negative) Catalase(positive)

3) Some strains {S.aereus) are coagulase test (positive) but some are coagulase (negative)

Several methods involving direct plating and or / enrichment techniques have been proposed for the detection of coagulase-positive Staphylococci in foods.

Testing methods for Staphvlococci

• Medium used for Staphylococci Baird-Parker agar medium.

• In this medium, its content all the nutrients that Staphylococci required

Presumptive test (more 100 Staphylococci)

• When greater than 100 Staphviococci aureus / gram are expected, direct plating of a diluted sample on

Baird-Parker medium followed by incubation for 30 hour at 37° C is recommended.

• Result: Staphylococci grow on this medium as a black colony surrounded by a clear zone

Confirmatory test

These tests include:

1) The utilization of glucose anaerobically, which separates the Staphylococci aureus from other species of Staphylococci.'

2) The ability to produce coagulase, which separatesStaphylococci aureus from other species of Staphylococci

How to carry out the coagulase test?

• The tests involve inoculation samples into blood serum and incubation for certain period under certain temperature to determine coagulation of the serum.

3) Also, S. aureus is separated from other Staphylococci based

on its property of utilizing mannitol anaerobically.

Presumptive test (low Staphvlococci aureus)

When count of less than 100 / Staphylococci aureus are expected, the most probable number (MPN) enrichment technique is recommended.

How to carry out the test?

Using 0.1g, 1g and 10g samples of the food to be analyzed.

Growth in anaerobic tellurite glycine mannitolpyruvate broth and incubated at 37°C is determined.

The positives tube are plated on Baird-Parker agar based medium and typical colonies are examined for the producing coagulase as described.

TESTING FOR PATHOGENIC BACTERIA

Pathogenic bacteria is a bacteria which have ability to cause infection to the organisms.

Example of pathogenic bacteria like Salmonella, Shigella and others.

Salmonella and Shigella are common pathogenic bacteria that cause food poisoning.

Detection of Salmonella in food

The detection method of Salmonella involves several stages:

a) Enrichment of samples

b) Plating of enrichment cultures

c) Screening

d) Confirmation test

Enrichment of sample

• The purpose to do enrichment of samples is to supply all the nutrients that required by Salmonella to grow.

How to carry out the enrichment method?

• The food sample are weighed and put into screw-capped jars.

• Nutrients broth was added into jars.•Then the lids of the jars are now tightened and the jars are

shaken vigorously after which they are incubated for 24 hours at 35°C.

Plating of enrichment cultures

After incubation a loopful of enrichment culture is streaked onto a plate of:

1) Salmonella-Shigella agar2) Brilliant green agar

The plates are then incubated for 22-24 hours at 35°C

Salmonella-Shigella agar: Salmonella colonies are usually colorless, transparent and usually larger than 5 mm in

diameter and may have black centers.

Brilliant green agar: Salmonella colonies are pink to deep red in color.

Screening

• From Salmonella-Shigella and Brilliant green agar, at least 5 typical colonies are selected and transferred in each case to a triple sugar iron agar slant.

• The slant being streaked and the butt stabbed.

• The slant are incubated overnight at 35° C and then examined.

• Result: For Salmonella positive, cultures the slant should be alkaline (purplish), the butt showed show acid (yellow).

Confirmation test

• Triple sugar iron agar positive cultures are now examined for their reaction to polyvalent antiserum.

• Result: Positive result showed a sign of agglutination with antiserum.

Testing for Shigella

The testing methods for Shigella involved

a) Enrichment of sample

b) Plating the enrichment culture

c) Screening test

d) Confirmation test

Enrichment test

The enrichment procedure for Shigella and the same are those for Salmonella

Plating the enrichment culture

After enrichment, the cultures were streaked on:

1) Salmonella-Shigella agar

2) Me Con key agar

The plate are incubated for 24hours at 35° C.

Result in broth agar: Typical colonies are colorless and transparent

Screening

Typical colonies are transferred into

1) Triple sugar iron slants, incubated at 35°C, at 24 to 48 hours.

Result: On this medium the slant is alkaline (purple) and the butt acid (yellow)

2) Lysine iron agar in which the slant is streaked and the butt stabbed

Result: The slant is alkaline (purple) and file butt of the lysine iron agar is acid (yellow)