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WATER
MICROBIOLOGY
Experiment 10
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
Rapid Kits
INTRODUCTION
Multiple Tube
Coliform Detection Techniques Fermentation Technique
Presumptive Test
Confirmatory Test
Completed Test
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)
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)
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
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 3
From positive tubes in
Prepare and sterilize EMBA confirmatory test, streak a Observe for colonies with
plates and NA slant lapful of the broth in EMBA metallic green sheen
plate
Tap Water
Tap Water
Tap Water
Pond Water
Pond Water
Pond Water
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 + + +
DSLB + + +
1ml SSLB - - -
0.1ml SSLB - - -
POND WATER
from PGH
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.