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Bacterial Enumeration

Gloria Phuong LeMicrobiology LabDr. Fran Norflus

Introduction

Bacteria divide via binary fission but not all daughter cells are viable

Bacterial enumeration is the measurement of bacterial cells per unit of sample (volume or mass)

Counting of bacterial cells is important in: Microbial ecology Epidemiology

Methods

Total count vs. viable count: all cells vs. viable cells capable of producing progeny

There are 2 ways of counting bacterial cells: direct and indirect Direct methods of enumeration involve counting

actual cells or colonies Indirect methods involve estimating the number of

cells based on cell mass, scattering of light through a culture (spectroscopy), or through statistical method called most probable number (MPN)

Methods

Method Enumeration Description

Indirect and viable MPN Statistical estimation based on growth patterns

Direct and viable Standard plate count Serial dilution of original sample and look for CFU

Indirect and total Spectroscopy Measure the transmittance of sample

Direct and total Staining and microscopy

Stain the cells with fluorescent dyes to make them visible in raw samples

Microbes are everywhere

Foods can cause hazards to a person’s health if They possess a large number of bacteria that can cause an

infection by mere ingestion of the food Contains microbial products of metabolism that can cause

intoxication of the body Food-handling techniques are important to prevent food

poisoning We will examine the presence of microbes in chopped beef

and chicken samples Normal and abnormal handling

Direct and viable method would be used

Procedures

Dilute the original sample (serial dilution)

Then use 0.1 ml of each dilution to perform the spread plate method (p. 56-59)

The plates will be incubated at 37oC for 48 hours

Spread Plate Method

Direct and viable count

After 48 hours, there will be colonies growing on the plates

We will perform standard plate count (direct method to determine viable cells)

Each colony originated from one mother cell We will count colony-forming-unit (CFU) Record in your notes either

TNTC: too numerous too count The exact CFU: 30-300

How to perform calculations?

First, we need to determine the concentrations of cells from countable plates

Then, we need to determine the dilution factor at each dilution (Df)

Next, we need to determine the total dilution (TD) TD=Df1xDf2xDf3…

Finally, to determine the concentration of cells in the original sample, we divide the number of CFU in the sample to the TD of that sample