Transcript Bacterial Enumeration

Bacterial
Enumeration
Gloria Phuong Le
Microbiology Lab
Dr. Fran Norflus
S
Introduction
S Bacteria divide via binary fission but not all daughter cells
are viable
S Bacterial enumeration is the measurement of bacterial cells
per unit of sample (volume or mass)
S Counting of bacterial cells is important in:
S Microbial ecology
S Epidemiology
Methods
S Total count vs. viable count: all cells vs. viable cells
capable of producing progeny
S There are 2 ways of counting bacterial cells: direct and
indirect
S Direct methods of enumeration involve counting actual
cells or colonies
S 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
S Foods can cause hazards to a person’s health if
S They possess a large number of bacteria that can cause an infection by
mere ingestion of the food
S Contains microbial products of metabolism that can cause
intoxication of the body
S Food-handling techniques are important to prevent food poisoning
S We will examine the presence of microbes in chopped beef and
chicken samples
S Normal and abnormal handling
S Direct and viable method would be used
Procedures
S Dilute the original
sample (serial dilution)
S Then use 0.1 ml of each
dilution to perform the
spread plate method (p.
56-59)
S The plates will be
incubated at 37oC for 48
hours
Spread Plate Method
Direct and viable count
S After 48 hours, there will be colonies growing on the plates
S We will perform standard plate count (direct method to
determine viable cells)
S Each colony originated from one mother cell
S We will count colony-forming-unit (CFU)
S Record in your notes either
S TNTC: too numerous too count
S The exact CFU: 30-300
How to perform calculations?
S First, we need to determine the concentrations of cells from
countable plates
S Then, we need to determine the dilution factor at each
dilution (Df)
S Next, we need to determine the total dilution (TD)
S TD=Df1xDf2xDf3…
S 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