This document describes the MPN (Most Probable Number) test for testing biological water quality and detecting the presence of fecal contamination and pathogens. It involves testing for the indicator organism E. coli using a multiple tube fermentation technique with three phases - presumptive, confirmed, and completed tests. Samples are collected and stored properly then inoculated into lactose broth tubes at serial dilutions and incubated to detect coliform growth. Positive tubes are then tested with BGLB/MacConkey broth and EC/A1 broth to confirm total and fecal coliforms, respectively. Definitive identification involves plating and gram staining from positive confirmed tubes.

1. MPN test

2. 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

3. • 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
Biological water quality testing

4. 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

5. 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

6. 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

7. 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
Sample collection,
preservation and storage

8. 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

9. Multiple-tube fermentation technique
Conducted in 3 phases
• Presumptive test
– Serial dilutions of a sample (to extinction) are incubated in
multiple tubes of lauryl tryptose 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)/MacConkey Broth at
35°C or in tubes of EC/A1 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

10. Multiple-tube fermentation technique
• Completed test
– Involves streaking of LES Endo agar plates with inoculum from
positive BGLB/MaCB or EC/A1 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

11. 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

12. 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

14. 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

15. 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, …

17. 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

18. 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)/MaCB for total coliforms
– Fermentation tubes with EC/A1 medium for fecal coliforms
Inoculate one BGLB/MaCB tube (and/or one EC/A1 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/MaCB and/or
EC/A1 tube with a 3 mm diameter sterile metal loop

19. Confirmed phase of the test
Incubate inoculated BGLB/MaCB tubes at 35±0.5°C
– Gas production within 48±3 hours of incubation is taken as
positive confirmed total coliform reaction
Incubate EC/A1 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/MaCB tubes give total coliform count
– Results adjusted on the basis of negative results with EC/A1
medium tubes give fecal coliform count

20. Completed test
Meant to definitively establish presence of coliform bacteria in the
positive confirmed tubes
Positive confirmed tubes of EC/A1 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/MaCB confirmed tube to obtain discrete colonies

22. 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

23. 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

24. 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

25. • 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
– Ammonium 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

26. • 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
Completed test

27. 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 MPN 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
considereddilutionlowesttheatsampleofmL
tablereadingMPN
mlMPNMPN
10
)100/(



28. 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
5/5-3/5-2/5-1/5 5-3-2-..
5/5-3/5-2/5-0/5 5-3-2-..

29. 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:
Precision of multiple tube fermentation test is low because of random
distribution and clustering of the coliform bacteria














tubestheall
insampleofmL
tubesnegative
insampleofmL
tubespositiveofNumber
mlMPN
100
100/

30. 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

31. 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

32. 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

33. 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

35. 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
The correct the calculated coliform count by multiplying with positive
verification percentage
filteredsampleofmL
countedcoloniesColiform
mLcoloniesColiform
100
100/



36. 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

37. 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

38. 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
Membrane filtration technique

39. 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)

40. 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

41. 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

42. 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

43. Lauryl tryptose broth
Tryptose 20 g
Lactose 5 g
K2HPO4 2.75 g
KH2PO4 2.75 g
NaCl 5 g
Sodium lauryl sulfate 0.1 g
Volume of medium 1 liter
pH after sterilization 6.8±0.2
Brilliant green lactose bile broth
Peptone 10 g
Lactose 10 g
Oxgall 20 g
Brilliant green 0.0133 g
Volume of medium 1 liter
pH after sterilization 7.2±0.2
Base ingredients of different media used
Lactose broth
Beef extract 3 g
Lactose 5 g
Peptone 5 g
Volume of medium 1 liter
pH after sterilization 6.9±0.2
EC Medium
Tryptose or trypticase 20 g
Lactose 5 g
Bile salts mixture or
bile salt no.-3
1.5 g
K2HPO4 4 g
KH2PO4 1.5 g
NaCl 5 g
Distilled water 1 liter
pH after sterilization 6.9±0.2

44. Nutrient Agar
peptone 5 g
Beef extract 3 g
Agar 15 g
Volume of medium 1 liter
pH after sterilization 6.8±0.2
LES Endo agar
Yeast extract 1.2 g
Casitone or trypticase 3.7 g
Thiopeptone or
thiotone
3.7 g
Tryptose 7.5 g
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

45. MacConkey broth
peptone 17 g
Proteose peptone 3 g
Lactose 10 g
Bile salts 1.5 g
NaCl 5 g
Neutral red 0.03 g
Crystal violet 0.001 g
Volume of medium 1 liter
A-1 broth
Lactose 5 g
Tryptone 20 g
NaCl 5 g
Salicin 0.5 g
Polyethylene glycol
p-isooctylphenyl ether
1.0 ml
Volume of medium 1 liter
pH adjustment 6.9±0.1
Add polyethylene glycol after heat
dissolving all solid ingredients
Base ingredients of different media used

46. LES Endo agar
Yeast extract 1.2 g
Casitone or trypticase 3.7 g
Thiopeptone or thiotone 3.7 g
Tryptose 7.5 g
Lactose 9.4 g
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
Distilled water with 20 ml/l of 95%
ethanol is used – controls background
growth and coliform colony size
Almost boil to dissolve agar but not
sterilize by autoclaving
Base ingredients of different media used
M- Endo agar
Tryptose and polypeptone 10 g
Casitone or trypticase 5 g
Thiopeptone or thiotone 5 g
Yeast extract 1.5 g
Sodium chloride 5 g
Lactose 12.5 g
K2HPO4 4.375 g
KH2PO4 1.375 g
Sodium desoxycholate 0.1 g
Sodium lauryl sulfate 0.05 g
Sodium sulfite 2.1 g
Basic fuchsin 1.05 g
Agar 15 g
Volume of medium 1 liter
Distilled water with 20 ml/l of 95%
ethanol is used – controls background
growth and coliform colony size
Almost boil to dissolve agar but not
sterilize by autoclaving

47. M-FC medium
Lactose 12.5 g
Tryptose or biosate 10 g
Proteose peptone No. 3
or polypeptone
5 g
Yeast extract 3 g
NaCl 5 g
Bile salt No. 3 or bile
salts mixture
1.5 g
Aniline blue 0.1 g
Volume 1 liter
Rehydrate in distilled water
containing 10 mL 1% rosolic acid in
0.2N NaOH.
Heat to near boiling and then
promptly cool to below 50C but do
not autoclave
M-VFC holding medium
Casitone, vitamin free 0.2 g
Sodium benzoate 4 g
sulfanilamide 0.5 g
Ethanol (95%) 10 ml
Distilled water 1 liter
Final pH 6.7
Heat dissolve the medium and sterilize by
filtration (pore size of filter 0.22µm)
LES MF holding medium
Tryptone 3 g
M-Endo broth MF 3 g
K2HPO4 3 g
Paraaminobenzoic acid 1.2 g
Agar 15 g
Distilled water 1 liter
Rehydrate in distilled water, heat to boiling to
dissolve agar and cool to 50C
Aseptically add 1 g of sodium benzoate, 1 g
of sulfanilamide and 0.5 g of cycloheximide
Base ingredients of
different media used