Biological water quality:

Coliform count

Dr. Akepati S. Reddy

Dept. Biotech. Environ. Sci.
Thapar University
 Biological water quality testing
Interest is to know about presence of waterborne pathogens
– Too many varieties to test and not feasible for direct methods
Presence and density of indicator organisms is established
Fecal contamination of water is established through testing for
the presence and density of an indicator organism
– Fecal matter of the infected is source for pathogens
– Fecal contamination indicates higher probability of pathogen
presence
Coliform bacteria (Escherichia coli), specifically fecal coliform is
the indicator organism
– It is present in water, whenever fecal contamination is there, in
larger numbers than any of the water borne pathogens
– Testing for its presence and density is cheaper, easier and faster
– Working with it does not produce serious health threats to
laboratory workers
 Biological water quality testing
• Actually tested for Total Coliform Count
– Since coliform can also be contributed by sources
other than fecal contamination, waters may also be
tested for Fecal Coliform Count
– Incubation temperatures are different (35C for total
coliform and 44.5C for fecal coliform)
• Two techniques are used to test waters for coliform
count
– Multiple tube fermentation technique
– Membrane filtration technique
 Sample collection,
preservation and storage
Cleaned, rinsed (final rinse with distilled water) and
sterilized (either by dry or wet heat) sampling bottles are
used
For collecting samples with residual chlorine, to prevent
continued bactericidal action, sodium thiosulfate is added
to sample bottles prior to sample collection
– 100 mg/l in case of wastewater samples
– 18 mg/l in case of drinking water
For collecting samples with high copper or zinc or high
heavy metals add chetaling agent EDTA to the bottle
prior to sterilization to give 372 mg/l in the sample
 Sample collection,
preservation and storage
Sample collection
– Use aseptic conditions
– Do not contaminate inner surface of stopper and bottle’s
neck and keep bottle closed untill to be filled with sample
– Fill without rinsing and replace stopper immediately
– Leave ample space (2.5 cm) to facilitate mixing by shaking
Sample collection from a tap
– Run the tap full for 2 to 3 min. to clear the pipeline, reduce
water flow to permit sample collection without splashing
– Avoid sampling from leaking taps
– Remove tap attachments (screen/splash guard!)
– If you desire clean tap tip with hypochlorite (100 mg/l), and
run it fully opened for 5-6 min prior to sample collection
 Sample collection,
preservation and storage
Sample collection from other sources
• In case of hand pump, run it for 5 min. prior to sampling
• In case of a well sterilized bottle can be fitted with weight
at the base and used
– Avoid contact with bed
• Avoid taking sample too near to banks or far from water
draw off point in case of river/lake/spring/shallow well
– If collecting from boat collect from upstream side
– Hold bottle near base, plunge it below water surface with
neck downward, turn it until its neck points slightly upwards
and mouth directed towards water current and collect
sample (if no current push bottle forward to create)
– Special apparatus can be used to mechanically remove
stopper under the water surface
 Sample collection,
preservation and storage

Start testing promptly

– If not to be started within 1 hr. ice cool the sample
Transport sample within 6 hr while holding temperature
<10C
– Use ice cooler for sample storage during transport
If testing not started within 2 hrs of receipt refrigerate
– Time elapsed between collection and testing should be <24
hrs
Record time elapsed and temperature of storage for each of
the samples analysed
 Multiple Tube Fermentation Test

Also known as MPN test (Most Probable Number)

• An estimate of mean density of coliforms - reported as
MPN/100 ml
• Poisson distribution (random dispersion) of coliforms is
assumed
Defintion of coliform bacteria for MPN test: All aerobic and
facultative anaerobic gram negative, non-spore forming,
rod shaped bacteria that ferment lactose with gas and
acid formation within 48 hrs at 35C
 Multiple-tube fermentation technique
Conducted in 3 phases
• Presumptive test
– Serial dilutions of a sample (to extinction) are incubated in
multiple tubes of lauryl triptose broth at 35°C for 48 hrs
– Positive results (production of gas/acid) is an indication for
the presence of coliforms
• Confirmed test
– Sample from positive tubes of presumptive test are
incubated in tubes of Brilliant Green Lactose Bile (BGLB)
or at 35°C or in tubes of EC broth at 44.5°C
– Positive result confirms presence of coliforms in case of
BGLB tubes and presence of fecal coliforms in case of EC
broth tubes
 Multiple-tube fermentation technique
• Completed test
– Involves streaking of LES Endo agar plates with inoculum
from positive BGLB/EC broth tubes for obtaining isolated
colonies
– Gram stain the cells from isolated colonies and examine
under microscope
– Gram negative, non-spore forming, rod shaped bacteria are
coliforms – completion test
• Calculation of MPN is
– Directly from poisson distribution
– From the MPN tables
– By Thomas equation
 Presumptive phase of MPN test
Lauryl tryptose broth or alternatively lactose broth is
used as medium
Dehydrated medium is mixed in distilled water, and heated
to dissolve the ingredients after pH adjustment
– Bromocresol purple (0.01 g/L) can be added for indicating
acid production
– Double strength medium is also required
– Quantity required depends on number of samples and
number of decimal dilutions
 Presumptive phase of MPN test
Medium is dispensed into fermentation tubes with inverted vials
(Derham tubes)
– Dispense double strength medium into the tubes that will
be inoculated with 10 ml sample to avoid dilution of
ingredients below the standard medium level
– Ensure that the medium level in the tubes is sufficient to
totally submerge the inverted vials
– 9 or 10 ml medium is usually dispensed into each tube
Close fermentation tubes with heat resistant caps and sterilize in
autoclave
 Presumptive phase of MPN test

Decimal dilution and inoculation of fermentation tubes

• Done in inoculation chambers aseptically and requires
– Sterilized dilution tubes each with 9 ml of dilution water
– Sterilized 1 ml and 10 ml capacity pipettes
Sterilized fermentation tubes with contamination free
medium and air bubble free inverted vials are used
– 3 or 5 fermentation tubes at each of the decimal dilutions
– One set of 3 or 5 tubes will be of double strength medium
 Presumptive phase of MPN test
Thoroughly mix the sample in sample bottle and aseptically
transfer 10 ml into each of the set of fermentation tubes
with double strength medium
– transfer 1 ml of the sample into a sterilized dilution tube
with 9 ml of dilution water
Thoroughly mix dilution tube contents and transfer 1 ml into
each of the 3-tube set with single strength medium
– transfer 1 ml of diluted sample from the dilution bottle into
the next dilution tube
Repeat the dilution and inoculation process till the desired
level of dilution is reached
– Dilution to extinction is the concept behind the decision
– Use a separate sterile pipette for each of the dilution
– Shake vigorously (samples & dilutions) while preparing
– Sample volumes used are 10, 1, 0.1, 0.01, 0.001, …
 Presumptive phase of MPN test
Mix fermentation tube contents after inoculation (through gentle
agitation) and incubate at 35±0.5C
After 24±2 hours of incubation shake each of the tubes gently
and examine for gas in the inverted vials or acidic growth
– If no gas or no acidic growth, reincubate and reexamine at
the end of 48±3 hours for gas or acidic growth
Record results (number of positive tubes for each dilution) and
submit positive tubes for confirmation phase of the test
– From recorded results read MPN value from MPN table
– If a positive tube of presumptive test gives negative result
in the confirmation phase accordingly adjust the results
 Confirmed phase of the test
Conducted on only the positive presumptive tubes
– If all tubes are positive at 2 or more dilutions, then conduct
the test on all the tubes of the highest dilution of positive
reaction and on all positive tubes of subsequent dilutions
Can be conducted simultaneously for both total coliforms and
fecal coliforms
– Fermentation tubes with Brilliant Green Lactose Bile Broth
(BGLB) for total coliforms
– Fermentation tubes with EC medium for fecal coliforms
Inoculate one BGLB tube (and/or one EC broth tube) from each
of the positive presumptive tubes
– Gently shake or rotate the positive tube of presumptive test
to resuspend microorganisms
– Transfer a loop full of the culture into the BGLB and/or EC
tube with a 3 mm diameter sterile metal loop
 Confirmed phase of the test
Incubate inoculated BGLB tubes at 35±0.5°C
– Gas production within 48±3 hours of incubation is taken as
positive confirmed total coliform reaction
Incubate EC broth tubes within 30 minutes of inoculation in water
bath at 44.5±0.2°C
– Immersed in the bath till medium level in the tubes is below
the water level in the water bath
– Gas production within 24±2 hours of incubation is taken as
a positive confirmed fecal coliform reaction
Adjust recorded results of the presumptive test if any of the
positive presumptive tubes gave negative reaction
– The results adjusted on the basis of negative results with
BGLB tubes give total coliform count
– Results adjusted on the basis of negative results with EC
medium tubes give fecal coliform count
 Completed test
Meant to definitively establish presence of coliform bacteria in
the positive confirmed tubes
Positive confirmed tubes of EC broth at elevated temperature do
not require completed test
– Positive confirmed tubes are taken as positive completed test
responses
Completed test involves
• Streaking one LES endo agar petriplate from each of the
positive BGLB confirmed tube to obtain discrete colonies
 Completed test
• Picking up a typical colony (or atypical colony) that is most
likely consist of coliform bacteria and transfering to
– A lauryl tryptose broth fermentation tube to check for gas
production on incubation at 35±0.5C for 24±2 hours
– A nutrient agar slant for incubating for 24 hours and
obtaining bacterial culture for Gram staining and
microscopic examination
• Microscopic examination of bacterial culture of the nutrient
agar slant after gram staining
Production of gas in the lauryl tryptose broth and demonstration
of gram negative, non-spore forming rod shaped bacteria are
taken as positive results
If the result is negative accordingly adjust the results recorded
during presumptive test
 Completed test
Liquify sterile LES endo agar, aseptically pour into sterile
petri plates and allow the poured medium to solidify
Gently shake or rotate the positive confirmed tube to
resuspend the organisms, take a loopful of the culture
and streak an LES endo agar plate
– Avoid picking up of any scum or floating membrane by the
inoculation loop
– Do streaking in such a way that isolated colonies obtained
Incubate the streaked plates at 35±0.5C for 24±2 hours
 Completed test
Bacterial colonies developed on the plate are divisible into
• Typical colonies: pink to dark red colonies with a green
metallic surface sheen (covering the entire colony, or
appearing only in a central area or on the periphery)
• Atypical colonies: pink, red, white or colourless colonies
without green metallic surface sheen
• Other colonies: non-coliform colonies
Pick up one or more typical colonies for inoculating the
secondary lauryl tryptose broth tubes and the nutrient
agar slants
– in the absence of typical colonies pick up the colonies that are
likely to contain coliforms
 Completed test
• Place a loopful of dilution water in the center of microscopic
slide and add to the water drop a loopful of the bacterial
culture of the nutrient agar slant
– Also maintain separate gram positive and gram negative control
cultures on the same microscopic slide for comparison
• Spread the culture in the water drop to make uniform
dispersion over an area of the slide, and then air dry & heat fix
• Stain the heat fixed smear with ammonium oxalate – crystal
violet solution for 1 min., rinse with tap water and drain off
– Amoonium oxalate – crystal violet solution: mix 2 g of crystal
violet, in 20 ml 95% ethyl alcohol, and 0.8 g ammonium oxalate,
in 80 ml distilled water, age for 24 hrs and filter
 Completed test
• Apply iodine solution for one min., rinse with tap water and
allow acetone alcohol solvent to flow across the smear till
colourless solvent starts flowing off from the slide
– Lugol’s solution (Iodine solution): Grind 1 g iodine crystals and 2
g KI in a mortar first dry then with distilled water till solution is
formed, and rinse the solution into amber bottle with 300 ml
distilled water
– Acetone-alcohol solvent: 1:1 mixer of 95% alcohol and acetone
• Counterstain the smear with safranin for 15 sec., rinse with
tap water, blot day and then examine microscopically
– Counterstain: dissolve 2.5 g safranin dye in 100 ml of 95% ethyl
alcohol and then add 10 to 100 ml distilled water
 Estimation of bacterial density
Estimated from the results of the presumptive phase of the test,
after necessary adjustments made consequent to the
negative results of confirmed phase and completed phase
Bacterial density is read from the MPX index table corresponding
to the number of positive tubes for 3 consecutive dilutions
– MPN index table for 5 tubes per dilution and the table for 3 tubes
per dilution are different
– MPN index table relates the number of positive tubes at 10, 1
and 0.1 ml sample volumes to MPN/100 mL
– When dilutions considered are different from 10, 1 and 0.1 ml,
for calculating MPN (from the index table reading) use
MPN reading table  10
MPN ( MPN / 100 ml ) 
mL of sample at the lowest dilution considered
 Estimation of bacterial density
When tested at sample volumes beyond 10, 1 and 0.1 ml,
choose the results of highest dilution (at which all the tubes
are positive) and the next two dilutions
5/5-5/5-2/5-0/5 ..-5-2-0
5/5-4/5-2/5-0/5 5-4-2-..
Of all the dilutions tested if only one gave positive results then
consider results of that dilution and of one dilution below and
one dilution above it
0/5-0/5-1/5-0/5-0/5 ..-0-1-0-..
If positive results are obtained even at a dilution beyond the
series of dilutions considered then add that positive result to
the results of the highest dilution considered
5/5-3/5-1/5-1/5 5-3-2-..
5/5-3/5-2/5-0/5 5-3-2-..
 Estimation of bacterial density
MPN index table do not include the unlikely combination of
results (the combination whose probability is <1%)
– Obtaining the unlikely combination of results usually indicates
faulty multiple tube fermentation technique
The MPN index table can also include 95% confidence limits
For estimating MPN from the unlikely combination of results and
from the results of a test where decimal dilutions are not used,
use the following (Thomas) equation:
Number of positive tubes 100
MPN / 100 ml 
mL of sample in  mL of sample in 
negative tubes   all the tubes 
   
Precision of multiple tube fermentation test is low because of
random distribution and clustering of the coliform bacteria
 MPN test for fecal coliforms
Elevated incubation temperature is used for the
separation of coliforms into those of coliform origin
and those of non-coliform origin
Two approaches can be followed
• Use of EC broth and incubation at 44.5±0.2C in the
confirmation phase of the test
• Use of a single step method with A-1 medium in place of
the three phase total coliform test
– EC medium is not recommended in place of A-1 medium –
prior enrichment in the presumptive medium is needed
– Inoculated tubes of A-1 broth need incubation first at
35±0.5C for 3 hours and then at 44.5±0.2C for 21±2
hours in a water both
– Gas production within 24 hours of incubation is a positive
reaction for fecal coliform
 Membrane filtration technique
Alternative to multiple tube fermentation technique
More precise, relatively more rapid and highly reproducible
technique
Relatively large volumes of sample can be tested and even
saline waters can be tested
Not good for waters with high turbidity and high in non-coliform
bacteria, and presence of toxic substances result in low
estimates
Results from membrane filtration are lower than from multiple
tube fermentation test due built in positive statistical bias
 Membrane filtration technique
Definition of coliform bacteria for membrane filtration
technique
– Aerobic and facultative anaerobic, gram negative, non-
spore-forming, rod shaped bacteria
– Bacteria that develop red colonies with metallic sheen
within 24 hrs of incubation at 35C on Endo-type medium
with lactose
– Pure cultures produce negative cytochrome oxidase
reaction and positive -galactosidase reaction
All red, pink, blue, white or colourless colonies (atypical
colonies) lacking metallic sheen are considered as non-
coliforms
 Membrane filtration technique
Measured volume of sample is filtered through a membrane
filter that completely retains coliform bacteria
– Duplicate volumes or quadruplicate volumes of a sample or a
few portions of a sample each of a different volume are also
often filtered for testing
Filter with coliforms is transferred to petri plates with LES
Endo agar or M Endo agar medium and inverted plates
with filter are incubated at 35±0.5C for 24 hours
– Filter can also be transferred to the surface of the absorbent pad
saturated with liquid medium and placed in a petri plate and
incubated
– For enrichment the filter can be incubated over an absorbent
pad saturated with lauryl tryptose broth for 1.5 to 2 hours at
35±0.5C in an atmosphere of 90% relative humidity prior to
incubation on endo medium for 20 to 22 hours
 Membrane filtration technique
After 24 hours of incubation count the number of coliform colonies
developed
– An ideal sample size is supposed to give about 50 coliform
colonies and <200 colonies of all types
– More than this number of colonies demand use of lesser volume
of the sample
– Smaller number of colonies need use of larger sample volume
From the number of colonies counted coliform count for the
sample is calculated by
Coliform colonies counted  100
Coliform colonies / 100 mL 
mL of sample filtered
The correct the calculated coliform count by multiplying with
positive verification percentage
 Membrane filtration technique
Coliform verification
• Necessary because typical metallic sheen colonies can often
be produced by non-coliform bacteria
• Verify 10% of the colonies or a minimum of 5 colonies or all the
metallic sheen colonies
• Can be by inoculating a lauryl tryptose broth tube with a
colony, incubating at 35±0.5C and observing for gas
production after 48 hours of incubation (gas production is a
positive test)
• Can be by cytochrome oxidase (CO) reaction test and by -
galactosidase (ONPG) reaction test – coliform reactions are
negative for CO and positive for ONPG
• Based on the verification the colony count the calculated
coliform count should be corrected
 Membrane filtration technique

Membrane filtration technique for fecal coliforms

• The filter is incubated on M-FC medium at 44.5±0.2C for
24±2 hours in water bath
• Fecal coliform colonies are various shades of blue
– Pale yellow colonies are atypical – verify these for gas
production in mannitol at 44.5C
– Non-fecal coliform colonies are gray to cream coloured
 Membrane filtration technique
Delayed incubation procedure
• Immediate performance of standard coliform test on the
collected sample may not always be feasible
• In such cases delayed incubation procedure is followed
– The sample is aseptically filtered immediately and the filter is
placed over a transport media for the transit till it is transferred to
the actual medium for standard testing
• Transport media are designed to keep the coliforms viable
and generally do not permit visible growth during transit time
– In case of total coliforms testing LES MF holding medium or M-
Endo preservative medium is used
– M-Endo medium after boiling to dissolve agar is cooled to below
50C and then 3.84 g/l of sodium benzoate is added to obtain M-
Endo preservative medium
– In case of fecal coliforms testing M-VFC holding medium is used
 Dilution water and peptone water
Distilled water or demineralized water used should be free from
traces of contaminating nutrients, dissolved metals, and
bactericidal or inhibitory compounds
Dilution water: Add 1.5 ml of stock phosphate buffer solution and
5 ml of magnesium chloride solution per liter of distilled water
and autoclave
– Stock phosphate buffer: Dissolve 34 g KH2PO4 in 500 ml
distilled water, adjust pH to 7.2±0.5 and makeup final volume to
one liter
– Dissolve 81.1 g of MgCl2.6H2O in distilled water and adjust final
volume to one liter
Peptone water: prepare 0.1% peptone solution from 10% stock
peptone solution, adjust pH to 6.8 and autoclave
Microbial suspensions in dilution water should not be maintained
beyond 30 min. (death or multiplication of bacteria can occur)
Culture media: Preparation and storage
Dehydrated media in the form of free flowing powders are
available
– Medium can also be prepared from its specified base ingredients
– Associated with the non-uniformity of composition
Dehydrated media stored in tightly closed bottles in dark low
humidity atmosphere at <30C is used
– Avoid using discoloured, caked and not-freely flowing media
– Use procured media (those containing sodium azide, bile salts or
derivatives, antibiotics, amino acids with sulfur) within 1 year
– After opening the bottle consume the medium within 6 months
Culture media: Preparation and storage
Rehydrate the medium and adjust pH to specified value
– Titrate small of the prepared medium to know the amount of acid
or alkali needed for pH adjustment
– Unless having buffering salts sterilization can reduce medium pH
by 0.1 to 0.3 units
– Overheating of a reconstituted medium can produce
unacceptable final pH
Dispense rehydrated medium into culture tubes within 2 hours
and sterilize
Sterilize in autoclave at 121C for 15 minutes
– Quickly cool the sterilized medium to avoid decomposition of
constituent sugars
– Avoid decomposition through sterilizing broths with sugars in 45
min cycle (use 121C for 12-15 min.)
– A-1 broth is sterilized at 121C for 10 min.
Follow manufacturer’s directions for the rehydration and
sterilization
Culture media: Preparation and storage
Use a prepared medium within one week
Do not store an unsterilized medium
• Fermentation tubes with medium can be stored at 25C
– Store out of direct sun light
– A-1 broth is stored in dark at room temp. for <7 days
– Avoid contamination and excessive evaporation (discard the
tubes with evaporation loss >1 ml)
• For storage beyond one week refrigerate
– Before use, keep refrigerated tubes overnight in incubator at
35C and discard contaminated tubes and tubes with bubbles
• Medium in screw capped tubes can be stored for 3 months
 Base ingredients of different media used
Brilliant green lactose bile broth
Lauryl tryptose broth
Peptone 10 g
Tryptose 20 g
Lactose 10 g
Lactose 5g
Oxgall 20 g
K2HPO4 2.75 g
Brilliant green 0.0133 g
KH2PO4 2.75 g
Volume of medium 1 liter
NaCl 5g
pH after sterilization 7.2±0.2
Sodium lauryl sulfate 0.1 g
Volume of medium 1 liter EC Medium
pH after sterilization 6.8±0.2 Tryptose or trypticase 20 g
Lactose 5g
Lactose broth
Bile salts mixture or bile 1.5 g
Beef extract 3g salt no.-3
Lactose 5g K2HPO4 4g
Peptone 5g KH2PO4 1.5 g
Volume of medium 1 liter NaCl 5g
pH after sterilization 6.9±0.2 Distilled water 1 liter
pH after sterilization 6.9±0.2
 Base ingredients of different media used
LES Endo agar Nutrient Agar
Yeast extract 1.2 g peptone 5g
Casitone or trypticase 3.7 g Beef extract 3g
Thiopeptone or thiotone 3.7 g Agar 15 g
Volume of medium 1 liter
Tryptose 7.5 g
pH after sterilization 6.8±0.2
K2HPO4 3.3 g
KH2PO4 1.0 g
NaCl 3.7 g
Sodium desoxycholate 0.1 g
Sodium lauryl sulfate 0.05 g
Sodium sulfite 1.6 g
Basic fuchsin 0.8 g
Agar 15 g
Volume of medium 1 liter
 Base ingredients of different media used
A-1 broth MacConkey broth
Lactose 5g peptone 17 g
Tryptone 20 g Proteose peptone 3g
NaCl 5g Lactose 10 g
Salicin 0.5 g Bile salts 1.5 g
Polyethylene glycol 1.0 ml NaCl 5g
p-isooctylphenyl ether Neutral red 0.03 g
Volume of medium 1 liter Crystal violet 0.001 g
pH adjustment 6.9±0.1 Volume of medium 1 liter
Add polyethylene glycol after heat
dissolving all solid ingredients
 Base ingredients of different media used
LES Endo agar M- Endo agar
Yeast extract 1.2 g Tryptose and polypeptone 10 g
Casitone or trypticase 3.7 g Casitone or trypticase 5g
Thiopeptone or thiotone 3.7 g Thiopeptone or thiotone 5g
Tryptose 7.5 g Yeast extract 1.5 g
Lactose 9.4 g Sodium chloride 5g
K2HPO4 3.3 g Lactose 12.5 g
KH2PO4 1.0 g K2HPO4 4.375 g
NaCl 3.7 g KH2PO4 1.375 g
Sodium desoxycholate 0.1 g Sodium desoxycholate 0.1 g
Sodium lauryl sulfate 0.05 g Sodium lauryl sulfate 0.05 g
Sodium sulfite 1.6 g Sodium sulfite 2.1 g
Basic fuchsin 0.8 g Basic fuchsin 1.05 g
Agar 15 g Agar 15 g
Volume of medium 1 liter Volume of medium 1 liter
Distilled water with 20 ml/l of 95% Distilled water with 20 ml/l of 95%
ethanol is used – controls background ethanol is used – controls background
growth and coliform colony size growth and coliform colony size
Almost boil to dissolve agar but not Almost boil to dissolve agar but not
sterilize by autoclaving sterilize by autoclaving
 M-VFC holding medium
Base ingredients of Casitone, vitamin free 0.2 g
different media used Sodium benzoate 4g
sulfanilamide 0.5 g
Ethanol (95%) 10 ml
M-FC medium
Distilled water 1 liter
Lactose 12.5 g
Tryptose or biosate 10 g Final pH 6.7
Proteose peptone No. 3 5g Heat dissolve the medium and sterilize by
or polypeptone filtration (pore size of filter 0.22µm)
Yeast extract 3g LES MF holding medium
NaCl 5g Tryptone 3g
Bile salt No. 3 or bile 1.5 g M-Endo broth MF 3g
salts mixture
K2HPO4 3g
Aniline blue 0.1 g
Paraaminobenzoic acid 1.2 g
Volume 1 liter
Agar 15 g
Rehydrate in distilled water
Distilled water 1 liter
containing 10 mL 1% rosolic acid in
0.2N NaOH. Rehydrate in distilled water, heat to boiling to
Heat to near boiling and then dissolve agar and cool to 50C
promptly cool to below 50C but do Aseptically add 1 g of sodium benzoate, 1 g
not autoclave of sulfanilamide and 0.5 g of cycloheximide
 Inoculation chamber
• Has working space with provisions for
– Outward flow of filtered bacteria free air
– Sterilization of the working space with UV radiation
– Sufficient lighting for working
– Bunsen burner flame
• Prior to use, clean the chamber and mop it with a sterilizing
agent and leave UV lights on for 15 minutes
• Use the chamber only after switching off the UV lights and
switching on of lights
• Maintain the chamber tidy and have only the minimum required
things within
• Make all transfers and inoculations in the heat zone of the
Bunsen flame