After reading this article you will learn about sampling for bacteriological examination.
Sampling Bottles- Samples for bacteriological examination shall be collected in clean, sterilized, narrow mouthed neutral glass bottles of 250, 500, or 1000 ml capacity. The bottle shall have a ground glass stopper having an overlapping rim. The stopper shall be relaxed by an intervening strip of paper between the stopper and the neck of the bottle.
ADVERTISEMENTS:
The stopper and the neck of the bottle shall be protected by paper or parchment cover. The bottle shall be sterilized in hot air oven at 160°C for one hour or an autoclave at 1.02 ± 0.03 kg/cm2 gauge pressure (15 ± 0.5 psi gauge pressure, 120°C temperature approximately) for 15 minutes. The sampling bottle shall not be opened except at time of sampling.
Note 1:
Discard bottles which have chips, cracks and etched surfaces. Before use, bottles should be thoroughly cleaned with detergent and hot water, followed by a hot water rinse to remove all traces of detergents. Then rinse them three times with laboratory pure water.
Sampling Procedure:
The samples shall be representative of the water to be tested and they should be collected with utmost care to ensure that no contamination occurs at the time of collection or prior to examination. The sample bottle shall not be opened till the time of filling.
The stopper and the neck of the bottle shall not be touched and they shall be protected from contamination. The bottle shall be held near the base, filled without rinsing, and the stopper replaced immediately. Then the brown paper wrapping should be tied to protect the samples from contamination.
Sampling from Taps:
Flame the tap (in case of plastic tap, apply alcohol or spirit, preferably rectified, and allow it to dry). The tap shall be opened fully and the water allowed to run to waste for two to three minutes or for a sufficient time to permit clearing of the service line.
ADVERTISEMENTS:
The flow from the tap shall then be restricted to permit filling the bottle without splashing. Leaking taps, which allow water to flow over the outside of the tap should be avoided as sampling points.
Note:
If the tap is connected to an overhead storage tank, this fact should be recorded in the sampling report.
Sampling Direct from a Source:
When the sample is to be collected directly from a stream, river, lake, reservoir, spring, or a shallow well, it shall be representative of the water that will be taken or supply to the consumers. Hence a sample shall not be taken or supply to the consumers. Hence a sample shall not be taken too far from a point of draw-off or too close.
ADVERTISEMENTS:
Areas of relative stagnation in a stream should be avoided. Samples from a river, stream, lake, or a reservoir can often be taken by holding the bottle in the hand near its base and plunging it neck downward, below the surface. The bottle shall then be turned until the neck points slightly upward, the mouth being directed against the current.
If no current exists, as in a reservoir, a current shall be artificially created by pushing the bottle horizontally forward in a direction away from the hand. If it is not possible to collect samples in this way, a weight may be attached to the base of the bottle which can developed into a separate colony on incubation.
Therefore, number, of colonies appearing on a plate does not necessarily represent the total number of organisms present in test volume. The results are expressed as number of colonies per ml.
Medium and Reagent:
Nutrient agar – Dissolve 1 g glucose, 5.0 g. of peptone and 3.0 g of beef extract in 1000 ml of distilled water. Adjust the pH to 7.2, distribute in required quantity and add 1.5 percent agar powder. Sterilize at 1.02 ± 0.03 kg/cm gauge pressure (15 ± 0.5 psi gauge pressure, 120°C temperature approximately) for 15 minutes in the autoclave.
Dilution Water:
ADVERTISEMENTS:
(a) Buffered dilution water:
To prepare stock phosphate buffer solution, dissolve 34 g of potassium dihydrogen phosphate (KH2PO4) in 500 ml of distilled water, adjust pH o 7.2 with sodium hydroxide solution (IN) and dilute to 1 litre with distilled water.
Add 1.25 ml of stock phosphate buffer solution to 1 litre of distilled water. Dispense in amounts that will provide 18 ± 0.4 ml or 9 ± 0.2 ml in 150×25 mm or 150 x 18 mm test tubes respectively. Sterilize in autoclave at 1.02 ± 0.03 kg/cm2 gauge pressure (15 ± 0.5 psi gauge pressure, 120°C temperature approximately) for 15 minutes.
(b) Quarter strength ringer’s solution:
Dissolve 9.00 g of sodium chloride, 0.42 g of potassium bicarbonate in 1 litre of water. This solution is known as Ringer’s solution. Dilute 500 ml. of this solution to 2 litre to obtain quarter strength Ringer’s solution.
Dispense in amounts that will provide 18 ± 0.4 ml or 9 ± 0.2 ml. in 150 x 25 mm or 150 x 18 mm test tubes respectively. Sterilize in autoclave at 1.02 ± 0.03 kg/cm2 gauge pressure (15 ± 0.5 psi gauge pressure, 120° temperature) for 15 minutes.
Preparation and dilution- Shake the samples about 25 times, withdraw required portion with a sterile pipette, and introduce into the petridish or dilution tube.
Plating:
Place 1.0 ml, or 1.0 ml of other suitable dilution to be used for plating in the petri dish first. Then add to the petri dish 10 to 15 ml of melted nutrient agar medium at a temperature of 43 to 45°C (tolerable to the skin).
The nutrient agar and the sample shall be thoroughly mixed over the bottom of the petri dish by tilting and rotating the dish several times, allow the plate to solidify and place immediately in the incubator in an inverted position.
Incubation:
Incubate the plates at 37°C for 24 hours.
Counting:
In preparing plates, plant such amounts of water for dilution which will give from 30 to 300 colonies on a plate. Always have two or more plates for each dilution. Report the result as the average of all plates falling within limits. It is not desirable to plant more than 1.0 ml in a plate. If the colonies are more than 300 or less than 30 from 1 ml sample, disregard it.
In practice, counts less than 30 occur when chlorinated water samples are plated. When the number of colonies is more than 300 in plate, report the count at ‘TNC’ (too numerous to count). Counting shall be done with an approved counting aid, such as colony counter. Record the number of colonies to the nearest 5 units per ml of report the temperature of incubations.
Test for Coliforms:
The coliform group includes all of the aerobic and facultative anerobic gram negative, non-spore forming rod shaped bacteria which ferment lactose with gas formation within 48 hours at 37°C. The standard test for the estimation of number of the coliform groups may be carried out either by the multiple tube dilutions test (presumptive test, confirmed test, or completed test) or by the membrane filter technique.
Multiple Tube Dilution Test (MTD):
The presumptive, confirmed and completed tests are presented as total independent procedures. In using these procedures, the worker must know what is to be the stage at which the test is to be ended, and details of the procedure throughout. Thus, if the worker knows that the test will be ended at the confirmed test, he will stop at the confirmed test stage only.
All the necessary information regarding the sample should be recorded. It is convenient to express the results of the examination of replicate tubes and dilutions in terms of most probable number. This term is actually an estimate based on certain probability formulae. The most satisfactory information is obtained when the largest portion examined shows no gas in all or a majority of the tubes.
The MPN value for a given sample is obtained by the use of MPN tables. Standard practice in water analysis is to plant fives tubes for each dilution and a minimum three different dilutions are employed. The results are to be recorded in the proper form. Table of MPN are given in Appendix B.
Media and Reagents:
(a) Dilution water
(b) MacConkey broth- This is used as a presumptive medium for the enumeration of coliform bacteria in water samples.
Its composition is as under:
In place of bile salt, which is a commercial product, sodium taurocholate or sodium tauroglycoholate may be used.
Dissolve all the ingredients and adjust the pH to 7.4. After adjusting the pH, add 1 ml of 1 percent alcoholic solution of bromocresol purple or 5 ml of 1 percent aqueous solution of neutral red. This will be the single strength medium.
Distribute 10 ml of the medium into 150 x 15 mm test tubes and add a Durham’s tube (25 x 5 mm) in an inverted position. Plug the tubes with non-absorbent cotton and sterilize at 115°C for 10 minutes in the autoclave.
This medium is used for 1 ml and the decimal dilutions of the water sample. For 10 ml. and larger aliquots a double strength medium is used. For the double strength medium add the above ingredients and double the quantities in 1000 ml of distilled water. This medium is dispensed into 10 ml. quantities in 150 x 18 mm test tubes added with Durham’s tube and sterilized.
(c) Brilliant Green bile lactose broth (BGB):
This medium is used as a confirmatory test for coliforms as well as for faecal coliforms. Its composition is as under:
Dissolve all the ingredients and adjust the pH to 7.4. Add 1.33 ml of 1 percent aqueous solution of brilliant green indicator. Distribute 4 ml quantities into 150 x 12 mm test tubes and add a Durham’s tube to each. After plugging with non- absorbent cotton, sterilize at 1.02 ± 0.03 kg/cm2 gauge pressure (15 ± 0.5 psi pressure, 120°C temperature approximately) for 15 minutes in the autoclave.
(d) Peptone water:
This is used for indole test or for preparing a liquid culture of an organisms. Its composition is as follows:
Dissolve all the ingredients. Adjust the pH to 7.4. Dispense 4 ml medium into 100 x 12 mm tubes and plug with non-absorbent cotton. Sterilize in the autoclave at 1.02 ± 0.03 kg/cm2 gauge pressure (15 ± 0.5 psi gauge pressure, 120°C temperature approximately) for 15 minutes.
(e) Mac Conkey agar:
The medium is used for the completed test or IMViC classification of coliforms. Its composition is as under:
Dissolve al the ingredients and adjust the pH to 7.4. Add 10 ml of 1 percent aqueous solution of neutral red indicator and 15 g or agar. Steam the medium for 15 to 30 minutes so that agar is dissolved properly and sterilize in autoclave at 1.02 ± 0.03 kg/cm2 gauge pressure (15 ± 0.5 psi gauge a pressure, 120°C temperature approximately) for 15 minutes.
After sterilization, cool to 45°C and prepare the plates by pouring 15 ml of melted agar per plate. Allow to solidify, invert and incubate at 37°C for drying as well as for sterility test.
(f) Nutrient agar slants:
Prepare the nutrient agar. Dispense while in the melted condition about 10 ml quantities into each tube (150 mm x 15 mm). Sterilize in the autoclave at 1.02 ± 0.3 kg/cm2 gauge pressure (15 ± 0.5 psi gauge pressure, 120°C temperature approximately) for 15 minutes.
After sterilization the slants are prepared by keeping the tubes to a solidify. Unless they are to be used, they should be stored in refrigerator.
(g) Kovac’s reagent:
It is used for indole test. Its composition is as under:
Dissolve paradimethyl aminobenzaldehyde in amyl alcohol and then add 25 ml of hydrochloric acid. The reagent shall be yellowish in colour. Store in amber coloured glass stoppered bottle.
(h) Gram staining reagents:
(a) Crystal violet is used as a primary stain.
(b) M-Coliform holding medium (LES holding medium) may be used as an alternative holding medium.
To prepare the medium, add the following reagents in 1 litre of laboratory pure water and mix (do not heat) to dissolve:
Procedure:
Saturate the sterile absorbent pads with about 2.0 ml of M-Endo Holding Medium or LES Holding Medium, prepared as outlined above. Pour off excess broth. Using a sterile forceps, place a membrane filter on the filter base, grid side up. Attach the funnel to the base of the filer unit; the membrane filter is now held between the funnel and base.
Shake the sample vigorously about 25 times and measure into the funnel with the vacuum off. If the sample is less than 10 ml, add 10 ml of sterile dilution water to the membrane filter before adding the sample. The sample volume should be such as to produce counts of 20-80 coliform colonies.
Filter the sample through the membrane and rinse the sides of the funnel walls at least twice with 20-30 ml sterile diluion water. Turn off the vaccum and remove the funnel from the base of the filter unit. With flame sterilized forceps remove the filter from the filter base and place grid side up on an absorbent pad saturated with M-Endo Holding Medium of LES Holding Medium, using a rolling action at one edge.
Exercise care to avoid trapping air bubbles under the membrane. Place top on petri dish and proceed with filtration of next volume. Clearly mark the lid of each petri dish, indicating location, and time of collection, time of incubation, sample number and sample volume. Use a water proof felt tip marker or grease pencil.
Inspect each membrane in the petri dish for uniform contact with the saturated pad. If air bubbles are present under the filter (indicated by bulges) remove filter with sterile forceps and roll into the absorbent pad again. Seal the petri dish by firmly pressing down the top.
Place the culture dish in shipping container and send it to the examining laboratory. At the examining laboratory remove the membrane from the holding medium. Place it in another dish containing M-Endo or agar medium and complete testing as already described.
Delayed Incubation Test for Faecal Coliforms:
The delayed incubation procedure for faecal coliforms is same as that for total coliforms except that M-VFC holding medium is used.
The composition of the medium is given below:
Preparation:
Add 4.7 g of medium (as given above) per litre of laboratory pure water containing 10 ml of 95 percent ethanol. Denatured alcohol should not be used. Heat slightly to dissolve the ingredients, then sterilize by membrane filtration (0.22 µm). Store prepared medium at 4°C. Discard after 1 month.
Test for Faecal Streptococci:
The test terms faecal streptococci and entercocci have been somewhat synonymously by many in recent years. The faecal streptococci group is indicators of faecal pollution of water because the general habitat of these organisms is the intestine of man and animals. They are gram positive cocci and ferment glucose with the production of acid only and are capable of growing in the presence of 40 percent bile and at 45°C.
On the basis of newer concepts of speciation of faecal streptococci, it is suggested that the terms faecal streptococci and Lancefields group D streptococeus be considered synonymous. The standard test for the estimation of number of the faecal streptococci may be carried out either by the multiple tube dilution technique or by the membrane filter technique.
Multiple Tube Dilution Technique:
Multiple tube dilution technique employs presumptive test procedures and confirmed test procedures.
Media:
(a) Dilution water
(b) Azide dextrose broth (ADB)-This is a presumptive test medium used for enumerating faecal streptococci in the water samples. Dissolve the following ingredients and adjust the pH to 7.3:
Dispense 6 to 7 ml of medium into 150 x 15 mm test tubes and plug with non- absorbent cotton. Sterilize in the autoclave at 1.02 ± 0, 0.03 kg/cm2 gauge pressure (15 ± psi gauge pressure, 120°C temperature approximately) for 15 minutes.
This is a single strength medium and used for 1 ml aliquots and decimal dilution when 10 ml sample or more has to be inoculated using double strength medium. This is prepared by using double the quantities given above in 1000 ml of water. 10 ml of this double strength medium is put into each 150 x 18 mm test tube.
(c) Ethyl violet azide broth (EVA):
Confirmatory medium used for enumerating faecal streptococci is as follows:
Dissolve all the ingredients and adjust the pH o 7.1. Add 1 ml of 0.083 percent alcoholic solution of ethyl violet. Dispense 10 ml medium into 150 x 18 mm test tubes and plug with non-absorbent cotton. Sterilize in the autoclave at 1.02 ± 0.03 kg/cm2 gauge pressure (15 ± 0.5 psi gauge pressure, 120°C temperature approximately) for 15 minutes.
Procedure:
Shake the water sample thoroughly before making dilution or before inoculation.
(a) Presumptive test:
Inoculate a series of tubes of azide dextrose broth with appropriate graduated quantities of the water to be tested (follow the same procedure as given for coliforms). Incubate inoculated tubes at 37°C. Examine each tube at the end of 24 hours for the presence of turbidity. If no definite turbidity is present reinoculate and read at the end of 48 hours.
(b) Confirmed test:
All azide dextrose broth tubes showing turbidity after 24 or 48 hours incubation must be subjected to the confirmed test. Transfer three loopfuls of growth from each azide dextrose broth to ethyl violet azide broth tubes. Incubate the inoculated tubes for 48 hours at 37°C.
The presence of streptococci is indicated by the formation of a purple button at the bottom of the tube, or occasionally by a dense turbidity. Find out the MP value from Appendix B. Record the results as numbers per 100 ml of the sample.
Membrane Filter Technique:
For outline of the method, description of the MF assembly, selection of the sample size and filtration of the sample, see ‘Standard methods’.
Medium:
Medium used for enumeration of faecal streptococci by membrane filter technique is known as M enterococcus agar and is given below:
Dissolve all the ingredients and adjust the pH to 7.2. Add 1 percent agar and heat sufficiently to dissolve agar. After slight cooling, add 1 ml of 1 percent sterile solution 2, 3, 5 triphenyl tetrazolium chloride per 100 ml of the medium. Pour the plate (15 mm x 60 mm) by adding 10 ml of the agar. Allow to solidify and use fresh plates.
Procedure:
Instead of pad, use a solid agar medium. Pour approximately 10 ml of M enterococcus agar into 60 mm petridishes. Allow to harden and place the filter on it. Inver and incubate at 37°C for 48 hours under humid chamber. Count all red and pink colonies with the help of stereomicroscope. Express the count as number of faecal streptococci per 100 ml of water.
Note:
Since the requirement of medium for each sample is very little, dehydrated powder medium is recommended.
Test for Clostridium Welchii:
General:
Clostridium Welchii are large rod-shaped, non-motile anaerobic bacteria which form spores which are relatively resistant to heat drying and ordinary bacterial agents.
Medium:
Litmus milk medium:
Keep fresh raw milk of low bacterial content in the refrigerator for 18 ± 1 hour so that the cream may separate. Remove the cream and add 10 percent litmus solution to the milk to give a purplish blue colour. Distribute in tubes in 10 ml quantities.
Add a mixture (melting point approximately 45°C) of equal parts of paraffin wax and petroleum jelly to form a layer about 2 to 5 mm thick of the surface of the medium. Steam for 30 minutes on 3 successive days. Test for sterility by incubation at 37.0° ± 0.5° for 48 ± 3 hours.