Biology 120 PreLab & POSTLAB

WATER
MICROBIOLOGY
Experiment 10

Jolaine Ashley T. Vallo

INTRODUCTION
 INTRODUCTION
Water Microbiology the study determines if the water is fit for
consumption, washing, bathing, irrigation,
conducted in the microbial for waterways and even for discharge to a
assessment of water body of water.

sewage
the scientific discipline that is concerned
with the study of all biological forms of potable water sources
microorganisms (bacteria, archaea,
viruses, fungi, parasites and protozoa) environmental fresh/marine water
that exist in water
domestic and agricultural waste water
 INTRODUCTION
Microorganisms in water
Numbers and types of bacteria present depend on:
Diverse
• Amounts of organic matter present
• Presence of toxic substances
• Water’s saline content
• Environmental factors such as pH, temperature,
and aeration

Performed to determine the

Total aerobic total aerobic microorganisms
plate count
 in water
Note: Drinking water often does not need to be diluted before surface plating
 INTRODUCTION
Water Quality Direct Test for Pathogens
the most important source of infection is water
Involve selective cultivation to large numbers
Common water borne diseases: • Time consuming
• Shigellosis (Shigella spp.) • Potentially dangerous to lab personnel
• Salmonellosis (Salmonella typhimurium) Molecular tests
• Gastroenteritis (Campylobacter spp.) • Require testing for each pathogen
• Cholera (Vibrio cholerae)
• Require expertise
• Giardiasis (Giardia lamblia)
• Cryptosporidiosis (Cryptosporium parvum)
Use of indicator organisms
Drinking Water Recreational Water Fish & wildlife habitat Indicators that water is contaminated with
No coliform pathogens
200 fecal coliforms/ 5000 fecal coliforms/
contamination
100ml 100ml
COLIFORMS
acceptable
 INTRODUCTION
Coliform Bacteria
Indicator organisms most widely used
indicator organism
•
•
•
Facultative aerobes
Gram-negative
Non-spore forming
• Rod-shape
Criteria of a good indicator for microbial water • Normally inhabit intestines
contamination is the • Should not be present in clean water
• Suitable for all water types coliform bacteria
• Ferment lactose with gas formation within 48 hours at
35oC

• Similar survival characteristics as

pathogens in water
• Present when pathogens are present
• Present in greater number than
pathogens
• Correlate with the degree of pollution
• Can be detected at low cost
• Non-pathogenic
 INTRODUCTION

Coliform Detection Techniques

Multiple Tube Fermentation Technique

Membrane Filtration Technique

Rapid Kits
 INTRODUCTION
Multiple Tube
Coliform Detection Techniques Fermentation Technique

Examination of fermentation of probable

present organism in multiple copies of media
in tubes. Presumptive Test
• Makes use of MPN for determination of bacterial
Confirmatory Test
population
• 95% confidence limit Completed Test
• Useful in low number of organism content
 INTRODUCTION
Multiple Tube
Coliform Detection Techniques Fermentation Technique

Presumptive Test

Lauryl Sulfate Tryptose Broth (LSTB) or

Lactose Broth (LB) with Durham tube is used.
Lactose-positive bacteria metabolize lactose with
gas formation within 24 hours or less =
presumptive evidence of coliform bacteria.
Durham tubes are for visualization of gas
production.
 INTRODUCTION
Multiple Tube
Coliform Detection Techniques Fermentation Technique

Confirmatory Test

Brilliant Green Bile Broth (BGLB) is used. It

contains two inhibitors of both gram- positive and
selected gram-negative organisms; i.e., oxgall and
brilliant green dye. Coliforms, which are resistant
to the action of the inhibitors and which ferment
the lactose, are able to replicate in this medium.
Fermentation is detected by gas production.
 INTRODUCTION
Multiple Tube
Coliform Detection Techniques Fermentation Technique

Completed Test

EMBA is a selective and differential medium used

in identification and isolation of Gram-negative
enteric rods. Lactose fermenters (i.e coliforms)
form colonies with dark centers and clear borders.
Non-lactose fermenters form completely colorless
colonies.
 INTRODUCTION
Membrane Filtration
Coliform Detection Techniques Technique

for the detection of stressed

total coliforms in treated The
drinking water and chlorinated standard
secondary or tertiary wastewater volume to
effluents be filtered
for drinking
not for wastewaters that have water
received only primary samples is
treatment followed by 100 mL
chlorination because of
turbidity in high volume
 INTRODUCTION
Coliform Detection Techniques
Colilert
Add reagent to sample and
incubate 24 hours. Read results:
Colorless = negative
Yellow = total coliform
Yellow/fluorescent = E. coli
Rapid Kits
Traps gas (seen as
Quantitrays Petrifilm bubbles) produced
Add reagent to sample. by coliforms.
Pour into Quanti-Tray Glucoronidase
(counts from 1-200) or indicator forms
blue precipitate
Quanti-Tray/2000 (counts around E.coli.
from 1-2419). Seal in
Selectivity and electivity
Quanti-Tray Sealer and BIORAD are based on the
incubate for 24 hours. detection of glucuronidase
and galactosidase
Read results: activities. Hydrolysis of
Yellow well = total chromogenic substrate
results in purple to pink
coliform E.coli colonies (gluc+/
gal+) and blue- green
Yellow/fluorescent wells
coliform colonies(gluc-/
= E.coli gal+).
OBJECTIVES
OBJECTIVES

This experiment aims to:

Perform and understand

1 microbiological test for water
potability

Understand the significance of the

2 different steps in potability testing

Use MPN table in qualifying amount

3 of indicator organism
METHODOLOGY
 METHODOLOGY

Sampling Coliform
Methods Detection
• tap water • Presumptive test
• from environment • Confirmatory test
• Completed test
 METHODOLOGY
Note: Add Sodium Thiosulfate to sample bottles before sterilization if the water
to be collected contains residual chlorine or other halogens added for
Sampling Methods: Tap Water disinfection. Add 0.1 ml of 10% sodium thiosulfate for every 100 ml of sample.
Add EDTA to sample bottles when water to be collected contains trace elements
greater than 10 ug/L.

1 2 3
Clean the tap and remove
Turn it on at maximum flow Open the tap again (this time
any attachments which may
rate for 1-2 minutes. Sterilize at medium flow rate) to get
cause splashing. Wipe the
with a flame for a minute. water sample at a bottle.
outlet with a clean cloth.

4 5
Leave a small air space in the bottle
Analyse water samples not more
to facilitate shaking at the time of
than 6 hours after sampling/24 hours
inoculation prior to analysis. Seal the
if chilled.
bottle tightly.
 METHODOLOGY
approx. 30cm below
Sampling Methods: Fresh Water
surface (1m deep)

1 Using a sterilized bottle,

2 Tilt bottle until neck
3
remove cap and quickly points slightly upward with
Leave a space of 2-3cm and
plunge the bottle upside the mouth directed towards
seal the bottle.
down to the required the current. Move bottle
sampling depth for the site. horizontally until filled.

4 5
Record time of sample collection Analyse water samples not more
and check sample identification than 6 hours after sampling/24 hours
labelling. if chilled.
 METHODOLOGY
approx. 15-20cm below
Sampling Methods: Marine Water
surface (0.5m deep)

1 Using a sterilized bottle,

2 Tilt bottle until neck
3
remove cap and quickly points slightly upward with
Leave a space of 2-3cm and
plunge the bottle upside the mouth directed towards
seal the bottle.
down to the required the current. Move bottle
sampling depth for the site. horizontally until filled.

4 5
Record time of sample collection Analyse water samples not more
and check sample identification than 6 hours after sampling/24 hours
labelling. if chilled.
 METHODOLOGY
*for turbid water, add 3 more SSLB
Coliform Detection: Presumptive Test that will be inoculated with 0.01 ml
sample water

1 2 3 Shake bottle of water

Prepare and sterilize: Label tubes: to be tested by inverting it 25
3 DSLB or DSLSTB and 3 DSLB with “10 ml” times. Aseptically transfer 10
6 SSLB* or SSLSTB 3 SSLB with “1 ml” ml water to DSLB tubes, 1
with durham tubes 3 SSLB with “0.1 ml” ml and 0.1 ml to SSLB

4 5
Incubate tubes for 24 hours at 35
oC. Observe and record number of Determine the presumptive MPN by
tubes with durham tubes filled with referring to the table in appendix
gas by 10% or more
 METHODOLOGY
Coliform Detection: Confirmatory Test

1 2 3
Inoculate lapful of broth
Prepare and sterilize BGLB Incubate tubes for 24-48
from positive tubes in
with durham tubes hours at 35 oC
presumptive test

4 5
Observe and record number of
tubes with durham tubes filled with Determine the confirmed MPN by
gas by 10% or more referring to the table in the Appendix
 METHODOLOGY
Coliform Detection: Completed Test

1 2
Prepare and sterilize EMBA
plates and NA slant
3
From positive tubes in
confirmatory test, streak a Observe for colonies with
lapful of the broth in EMBA metallic green sheen
plate

4 5Perform gram stain on bacteria in NA

Select an isolated colony with slant. Formation of metallic green sheen
metallic green sheen and streak in of a gram-negative non-spore forming
NA slant and incubate for 24 hours bacteria that can ferment lactose is a
completed positive test for coliform
RESULTS
 RESULTS

Tap Water

Presumptive Test: DSLB

Observations/Interpretation:
No bubbles for all tubes
Negative
 RESULTS

Tap Water

Presumptive Test: 1ml SSLB

Observations/Interpretation:
No bubbles for all tubes
Negative
 RESULTS

Tap Water

Presumptive Test: .1ml SSLB

Observations/Interpretation:
No bubbles for all tubes
Negative
 RESULTS

Pond Water

Presumptive Test: DSLB

Observations/Interpretation:
All tubes with bubbles
Positive
 RESULTS

Pond Water

Presumptive Test: 1ml SSLB

Observations/Interpretation:
All tubes with bubbles
Positive
 RESULTS

Pond Water

Presumptive Test: .1ml SSLB

Observations/Interpretation:
All tubes with bubbles
Positive
 RESULTS
Confirmatory Test

DSLB 1ml SSLB 0.1ml SSLB

All tubes with bubbles No bubbles for all tubes No bubbles for all tubes
Positive Negative Negative
RESULTS
Completed Test

Observations/Interpretation:
All three segments
yielded green-metallic
sheen colonies.
Positive
DISCUSSION
 DISCUSSION
Media components and functions
Tryptose provides the nitrogen, carbon compounds, vitamins and amino acids
Lactose is the fermentable sugar
LSTB/ Sodium lauryl sulfate inhibits organisms other than coliforms

LB Bile salts inhibit gram-positive bacteria especially bacilli and faecal Streptococci
Sodium chloride maintains the osmotic balance of the medium
Potassium phosphate controIs the pH during fermentation of lactose

BGLB contains two inhibitors of both gram- positive and selected gram-negative
BGLB organisms; i.e., oxgall and brilliant green dye

Differential basis of EMBA involves two indicator dyes, eosin and methylene blue,
EMBA that distinguish between lactose fermenting and non-lactose fermenting organisms
 DISCUSSION
The Most Probable Number Method
MPN Tables
enables us to
calculate for a
sample the
microbial
numbers that
are statistically
likely to lead
such a result.
 DISCUSSION
The Most Probable Number Method
Presumptive Test
TAP WATER POND WATER
from the 4th floor GAB comfort room from PGH
Tube 1 Tube 2 Tube 3 Tube 1 Tube 2 Tube 3

DSLB - - - DSLB + + +

1ml SSLB - - - 1ml SSLB + + +

0.1ml SSLB - - - 0.1ml SSLB + + +

Positives: 0-0-0 Positives: 3-3-3

Indication: <3.0 MPN per ml Indication: >1100 MPN per ml
95% of the water samples that give this result contain 95% of the water samples that give this result contain 420-
0-9.5 bacteria, with <3 being the most probable number. ∞ bacteria, with >1100 being the most probable number.
 DISCUSSION
The Most Probable Number Method
Confirmatory Test
POND WATER
from PGH
Tube 1 Tube 2 Tube 3

DSLB + + +

1ml SSLB - - -

0.1ml SSLB - - -

Positives: 1-1-1 Indication: 11 MPN per ml

95% of the water samples that give this result contain
3.6-38 bacteria, with 11 being the most probable number.
 DISCUSSION
The Most Probable Number Method
Completed Test

POND WATER
from PGH

Segment 1 Segment 2 Segment 3

green metallic green metallic green metallic

DSLB
sheen sheen sheen

Indication: Positive for coliform

 DISCUSSION
Coliform Calculation

For liquid samples For solid samples

like water: diluted in liquid:
coliform count Fecal coliforms/ = colonies counted
Coliform/ml = (dilution chosen) x (% dry solids)
sample filtered gram dry weight

*desired range of 20 to 60 fecal

coliform colonies
 DISCUSSION
Coliform Calculation Sample Problem 1

100 ml of water from a chlorine treated swimming

pool was tested for presence of coliform by
membrane filtration. 110 colonies were seen to
grow on the medium. How do you report for the
coliform count of the water in swimming pool?
Answer: 11 coliforms/10 ml or 1coliform/ml
 DISCUSSION
Coliform Calculation Sample Problem 2

There were 22 colonies observed on the 1:10 000

dilution plate of a biosolid with 4% dry solids.
Fecal coliforms/ = colonies counted
(dilution chosen) x (% dry solids)
gram dry weight

Fecal coliforms/ = ________22________ = 5.5 x 10 6

gram dry weight (0.0001) x (0.04) fecal coliform/g

dry weight
 DISCUSSION
Coliform Calculation Sample Problem 3

If no filter has a coliform count falling in the ideal range (20 to 60), total the coliform
counts on all countable filters and report as fecal coliforms per gram dry weight.

There were 18 colonies observed on the 1:10 000 dilution

plate and 2 colonies observed on the 1:100 000 dilution
plate of a biosolid sample with 4% dry solids.
Fecal coliforms/ = colonies counted
(dilution chosen) x (% dry solids)
gram dry weight
Fecal coliforms/ _________18 + 2__________
= = 4.5 x 106 fecal coliform/g
gram dry weight (0.0001+0.00001) x (0.04) dry weight
GUIDE QUESTIONS
 GUIDE QUESTIONS
1. What are the reasons for using the different media in
each step of the test?

Different media are used for the specificity and

enrichment of coliform.
• DSLB/SSLB: Lactose-fermenting bacteria
• BGLB: Specific for coliform
• EMBA: Visual/microscopic evidence of coliform
 GUIDE QUESTIONS
2. Is MPN a qualitative of quantitative means to
determine the potability of water. Why?
MPN is a qualitative means to determine the potability of
water. It relies on the diagnostic characteristics of the
samples (gas production, turbidity). Most of the time, water
samples to be tested are heterogeneous making it impossible
to determine the exact cell numbers of individual organisms.
The technique does not rely on quantitative assessment of
individual cells, instead it relies on specific qualitative
attributes of the microorganism being counted.
 GUIDE QUESTIONS
3. Give several procedures that are used to reduce
microbial load in water supplies.

Some ways by which we can reduce microbial load in

water supplies include:
• Deionization
• Ozone-filtration
• Chlorination
• UV-light radiation
CONCLUSION
 CONCLUSION

E. coli and other types of coliforms

are present in significant amounts
in the water sample obtained from
the PGH pond water; while being
in negligible to zero amounts in the
4th floor GAB comfort room faucet.