Microbio Lab Transes (mod 7-11) 1.

Serial dilution

Module 7: Isolation Techniques

Bacteria exist in mixed populations

In order to adequately study and characterize an

individual bacterial species, one needs a pure culture.

Pure Culture – Bacterial suspension consisting of a single

species.

Techniques

1. Streak Plating – mixed culture is spread on a solid agar 2. Direct count – Greater number of cells
plate using a wire loop; best method for isolation of because dead cells are also counted (aside
colonies. from the living cells)

2. Spread Plating – mixed culture is spread on a solid agar

plate using a L –rod; used for colony counting

3. Pour Plating – mixed medium is incorporated with a

molten agar then poured into a plate; best for cultivating
anaerobic microbes.

Module 8: Estimating Bacterial Counts

Microbial Growth Dilution= vi/vf

Dilution Factor = vf/vi
 Increase in the number of cells
Cells = (average # of cells in 5 squares) (1/4
 Dependent on the nutrients of the culture
nl) (Dilution factor) (10^6)
medium and the environmental requirements of
3. Viable count – Colony forming units are only
the microorganisms.
considered;
Growth Curve represent
viable cells

Formula

# of
colonies DF

Microbial Counts Volume

plated

 Used to determine how microorganisms grow,

assess the foods and monitor industrial process.
 Also an indicator of spoilage.

Procedure
Pour plate method  Faecal coliform count
 Total coliform count
 Anaerobic to Facultative bacteria will grow.
 Faecal streptococci
 Volume plated is always 1 ml to 0.1 ml
 Cyst/ova of parasites
 Usually lower count than Spread Plating because
of the hot medium poured into the bacterial Water Quality
suspension
 Drinking water – water intended for direct
Spread plate method human consumption or use in food preparation.
 Where high quality waters are scarce, the quality
 Aerobic to Facultative Anaerobe will grow.
of water used for other domestic purposes need
 Volume plated is always 0.1 ml.
not be as high as that of drinking water.
 Measuring the size of microbial population provides
 Potable water – water suitable (both health and
valuable information about the biology of
acceptability considerations) for drinking and
microorganisms.
cooking purposes.
 In microbial counts, two methods are being used.
 Other categories for recreational, marine,
The direct count and the viable count.
coastal
 Viable count is more reliable because the observer
will only consider the living cells whereas in direct Guidelines in Identifying Priority Drinking-Water
count both living and dead cells are counted. Quality Parameters for Monitoring

Based on its health significance and acceptability, the

 Spread plating (viable count) can be used to
following priority parameters shall be tested:
measure aerobic to facultative anaerobes while
pour plating measures anaerobic to facultative  Microbiological  Turbidity
anaerobes.  Arsenic  Iron
 It is possible that pour plating method will give a  Cadmium  Ph
lower count because the melted medium can kill  Lead  Manganese
the cells.  Nitrate  Chloride
 Benzene  Sulfate
Module 11: Bacteriological Analysis of Water  Color  TDS
Water Quality Parameters

Physical Microbiological Quality of Water: Coliform Detection

 Turbidity  Standard Water Analysis (Presumptive - Multiple

 Palatability Fermentation tubes, Confirmatory, and
Completed)
 Conductivity
 Membrane filtration techniques - alternative
 Total dissolved solutes
 Purposes:
 Organoleptic properties
o Quality testing of water to ensure
 Etc.
whether the water is safe or not in terms
Chemical of bacteria present in it.
o A group of bacteria referred to as faecal
 pH
coliforms act as an indicator of faecal
 Dissolved oxygen concentration contamination of water.
 Residual free chlorine o The presence of large numbers of fecal
 Radionuclides coliform bacteria would indicate a very
 Organic/ inorganic chemical contents high probability that the water could
 Etc. contain disease‑producing organisms
Microbial making the water unsafe for
consumption
Classification of Enterobacteriaceae:  a statistical method of determining microbial
populations - total coliform.
 Uses multiple dilution tube technique
 Resultant number coding is translated by
mathematical probability tables into MPN/100
ml.
 indicates the presence or absence of an
approximate number of coliform
 Test use for Presumptive

Coliform Organisms:

Characteristics Detection

rod-shaped, non-spore- By gram staining or

forming gram-negative
Using selective medium
bacteria
for g-neg i.e EMB, Endo
agar, MacConkey

Ferment lactose at 37oC. Gas production caught in

the Durham tubes; dark
colonies pink, red, purple
with green metallic sheen.

production of acid Acid pH cause yellow

coloration in Lactose
broth; metallic sheen and
purple surrounding with
low pH on plates.

Acetaldehyde Dark coloration of

colonies.

Water Quality Standards

Fecal Coliforms: Microbiological standard
 subgroup of coliform bacteria that has a high To frame the results within the Philippine
positive correlation with fecal contamination context, the National Standards for Drinking Water
associated with all warm-blooded animals. states that, using standard methods of analysis (DOH,
 These organisms are rapid lactose fermenters. at
44.5oC.
 Fecal indicator organisms – microorganisms that
when detected present in water supply signals
fecal pollution of water Philippines National Standards for Drinking Water,
2007):
Most Probable Number (MPN)
  Total coliforms should be at a conformity risk Location Before rehabilitation After rehabilitation
level (1 cfu/100mL) for 95% of samples taken in of Water 2019 2020
Sampling
a given time period (defined based on sample
location).
 E.coli test must give a result of <1.1 MPN/100mL
Fecal Coliform MPN/100 ml
The code on sanitation (DOH, 1995) states that water
Padre 7.21 M 920,000
should not be supplied for public use unless a level of
Faura
treatment has been provided based on the following
Across 35 M 11 M
Water quality results from coliform organisms. Aristocrat
Water Quality Results Yatch Club 110 M 54 M
(MPN/100mL) WHO Risk Treatment
Levels Target for 110
Water sources that fall in Manila Bay
this category are (Class SB
<50 MPN/100mL Coastal
characterized as ”low
Conformity, Low and and
degree of contamination”
intermediate risk levels Marine
and requires disinfection
Water)
alone
Water sources that fall in
>50 MPN/100mL and this category are
<5,000 MPN/100mL characterized as ”high
High and very high risk degree of contamination”
level and requires “complete
treatment”
WHO Risk Level corresponding to E. coli level in sample
adapted from WHO (1997) by changing thrmotolerant
coliform to E.coli (Doyle & Erickson, 2006) (Metcalf,
2006).
E.coli in samples
Risk Level
(CFU/100 mL)
Conformity <1
Low 1-10 II. Confirmatory
Intermediate 10 -100
 To confirm if coliform ferments the lactose,
High 100 -1000
hence, producing the gas and not the other
Very high >1000
microbes.
 Brilliant green lactose broth –
o Bile is inhibitory to gram-positive
microorganisms, while brilliant green
dye inhibits selected gram-negative
bacilli.
o Lactose-fermenting organisms resistant
to these inhibitors are detected by the
production of gas. Gas production is
noted by the appearance of bubbles in
Manila Bay’s Water Quality Improving One Year After the Durham tube.
Rehab Started—Denr Jan. 2020

III. Completed test

  Uses selective and differential medium such as Green Metallic sheen: Gram neg,
EMB. (E. coli) Darkened purple
with strong acids
Observation on Interpretation Remarks
produced from
EMB plate
lactose
E. coli only Positive Fecal Metallic green with
coliform low pH – rapid
fermenter
Both E. coli and Positive With Fecal Fecal coliform
coliform
E. aerogenes Dark pink/red
(Enterobacter Gram neg,
E. aerogenes only Negative E. aerogenes Darkened with
is widely aerogenes)
strong acids
distributed in produced from
nature lactose
outside of the No Metallic green,
intestinal pH did not lower –
tract. slow fermented
Coliform, but non-
Eosin Methylene Blue Agar fecal

 Selective, differential agar medium used for

isolation of gram-negative rods in a variety of Light pink/Colorless ( Gram neg,
specimen types. non-fermenters/non- Did not darken - no
 Methylene blue inhibits the gram + bacteria coliform, acids produced
(eosin to a lesser extent); while eosin changes P. aeruginosa) from lactose
No Metallic green,
color to a dark purple, when the medium around
pH did not lower –
the colony becomes very acidic.
non-fermenter
 Lactose – key nutrient; fermented by coliforms Non-coliform
to produce acid
Membrane filtration Technique – alternative to MPN

 Is a technique that uses a physical barrier

(usually a porous membrane or filter) to
separate particles and microorganisms
suspended in a fluid sample including food, air
and water.
 Cellulose nitrate and cellulose acetate filter
membranes retain particles or microorganisms
larger than their pore size primarily by surface
capture.
 Estimates bacteria and other microbes present
in public health samples such as water, air and
food.
Expected Results on plates
Membrane filtration Technique – alternative to MPN

 major advantage of the membrane filtration

technique over the MPN technique is that it
isolates discrete colonies of bacteria whereas
MPN only indicates the presence or absence of
 an approximate number of microorganisms in a  Preparation time is reduced when compared to
given sample other traditional methods of estimating bacteria
 Bacteria trapped on the filter will grow into such as the MPN technique.
visible colonies that can be counted (CFU/100  Membrane filtration techniques provide results
ml) on bacterial count within 24 hours unlike the
 Uses Endo agar or EMB MPN technique that takes several days to
conclude.

Figure 1: Nitrocellulose membrane with 0.45 um pore Figure 4: Bacterial colonies growing on membrane
size. A grid is printed on the membrane to assist with filter inoculated on agar plate.
counting bacterial colonies after incubation.

Figure 2: Bacterial count using the membrane filtration

technique. (A) A known quantity of the test sample (1) is
filtered through a membrane filter (2). (B-C) The
membrane filter is placed onto a Petri plate using sterile
forceps. (D-E) After the adequate incubation period,
colony forming unit (CFU) values on the surface of the
filter can be counted.

Advantages of Membrane Filtration Technique

 Fast and simple way to estimate bacterial

population in a sample.
 Large sample volumes can be tested using the
membrane filtration technique.