Measuring Microbial Growth - Overview

Direct Methods
• Plate counts Indirect Methods
• Filtration • Turbidity
• MPN • Metabolic activity
• Direct microscopic count • Dry weight
Direct Measurements
of Microbial Growth
1. Plate Counts

Serial Dilutions
Kultivasi Mikroba
Pertitungan dan Kurva tumbuh bakteri

Result : Growth curve

2. Membrane Filtration
Advantages
1. sensitive (theoretically, a single cell can be detected),
2. allows for inspection and positive identification of
the organism counted.

Disadvantages
1. only living cells develop colonies that are counted;
2. clumps or chains of cells develop into a single
colony;
3. colonies develop only from those organisms for
which the cultural conditions are suitable for growth.
Detection of microorganisms in water
• Detection techniques
• Multiple tube fermentation or MPN method
• Membrane filtration method
• Indicator and index microorganisms
• Indicator organisms indicate that water received
contamination of intestinal origin.
• Coliform
• faecal coliforms
• E. coli
Coliforms
• Most of these organisms are members of the normal flora of humans
and/or animals and are considered opportunistic pathogens.

• Most are found in the colon. Most of these organisms possess

fimbriae that is used as appendages for adhesion purposes.
Coliforms
• Coliforms- refers to the various genera of the family
Enterobacteriaceae which are commonly found as
contaminants in water.
• Coliform bacteria are described as:
▪ Rod-shaped bacteria, gram-negative, non-spore
forming, facultative anaerobes
▪ Ferment lactose, producing gas and acid within 48
hours when cultured at 35oC.
▪ Their lack of ability to form spores makes them
more susceptible to destruction by environmental
conditions.
• This group are referred to as "coliforms"
because they share similar morphological and
biochemical characteristics.

• Coliform bacteria: organisms that are under

the genus Escherichia, Enterobacter,
Klebsiella, Serratia, Citrobacter.
 What are fecal coliform bacteria?

▪ are bacteria found in feces.

▪ normally reside in the intestinal tract of warm-blooded
animals. Outside of a warm-blooded host, fecal
coliforms are short-lived compared to the coliform
bacteria that are free-living
▪ The fecal category contains both pathogen (disease-
causing) and nonpathogenic bacteria.
▪ An example is Escherichia coli.
▪ The presence of fecal coliforms is indicative of fecal
contamination and of the potential presence of enteric
pathogens (disease causing organisms which originate
in the digestive system), especially bacterial pathogens.
E.coli
• E. coli are found in intestine, their ability to survive for
brief periods outside the body makes them an ideal
indicator organism to test environmental samples for
fecal contamination.
• E. coli are Gram negative bacterium that is
commonly found in the lower intestine of warm-
blooded animals,
while other coliforms( Enterobacter, Klebsiella) can
be found on plants and in soil.
3. Most Probable Number

• Multiple tube MPN test

• Count positive tubes
• Performed in 3 steps
1. Presumptive test
2. Confirmatory test
3. Completed test
1. Presumptive test
• Compare with a statistical table
10 mL 1 mL 0,1 mL
2. Confirmatory Test

BGLB (Brilliant Green Lactose Bile) components

 Peptone: a source of nitrogen, vitamins and
minerals.
 Lactose: fermentable carbon source
 Oxgall (bile) and brilliant green: inhibitor of gram-
positive bacteria and most gram-negative bacteria
except coliforms
 Basic fuchsin and erioglaucine: pH indicators
 Monopotassium phosphate: buffering agent.
BGLB medium

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Confirmatory Test

E. coli vs E. aerogenes
3. Completed Test

From each of the solid-medium plates used for the

confirmed test, transfer selected colonies each to:
▪ Lactose broth medium , and incubated for 35°C for
24hours (look for gas production)
▪ Culture on NA slant. The agar slants should be
incubated at 37°C for 24± 2 hours
▪ Perform gram-staining of cultures from the slants
and examined microscopically.
Flow chart of Confirmed and Completed MPN
 Most Probable Number

Advantages Disadvantages
 Relatively simple and  Time consuming and
sensitive labor intensive
 Can count a specific  Requires large
type in the presence of volumes of glassware
others  Doesn’t give the
 Can use large sample “real” value
volumes
4. Direct Microscopic Count

• Need a microscope, special slides, high power

objective lens
• Typically only counting total microbe numbers, but
differential counts can also be done
Indirect Measurements
of Microbial Growth
1. Turbidity measurements

• Cells act like large particles that scatter visible light

• A spectrophotometer sends a beam of visible light through a
culture and measures how much light is scattered
• Scales read in either absorbance or % transmission
• Measures both live and dead cells
• Indirect but very rapid and useful method of measuring
microbial growth.

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▪ another indirect way of estimating bacterial numbers is
measuring the metabolic activity of the population (for
example, acid production or oxygen consumption).

▪ For filamentous organisms such as fungi, measuring dry

weight is a convenient method of growth measurement.

▪ You won't know how the measurement of turbidity with a

spectrophotometer or measurement of metabolic activity or
any other indirect measurement correlates to cell number
unless you do a standard curve.
Table 1. Some Methods used to measure bacterial growth
Method Application Comments
Enumeration of bacteria in Cannot distinguish living from
Direct microscopic count
milk or cellular vaccines nonliving cells
Enumeration of bacteria in
Viable cell count (colony Very sensitive if plating
milk, foods, soil, water,
counts) conditions are optimal
laboratory cultures, etc.
Estimations of large numbers Fast and nondestructive, but
Turbidity measurement of bacteria in clear liquid cannot detect cell densities
media and broths less than 107 cells per ml
Measurement of total cell
Measurement of total N or only practical application is in
yield from very dense
protein the research laboratory
cultures
Measurement of Biochemical
Requires a fixed standard to
activity e.g. O2 uptake CO2
Microbiological assays relate chemical activity to cell
production, ATP production,
mass and/or cell numbers
etc.
Measurement of dry weight
probably more sensitive than
or wet weight of cells or Measurement of total cell
total N or total protein
volume of cells after yield in cultures
measurements
centrifugation