Isolation of Bacteria | Bacterial Enumeration

Isolation of Bacteria | Bacterial Enumeration

In the environment, mixed population of bacteria live in very large numbers. Identification of them is often required. To identify usually they have to be grown in the laboratory separately. This is called isolation. There are several easy methods for isolation of bacteria.

Streak plate method

The streak plate method is a technique to isolate pure strain of microorganisms (mostly bacteria), from a mixed population. After incubation period individual bacterial cells are separated and well-spaced from each other. Then pure culture should be reinoculated in a new plate. This is only an isolation method, not an enumeration method. The streak plate technique gradually dilutes the number of bacteria in each “quadrant” of the plate. So, the last quadrant should have small, isolated colonies that can be easily studies.  By using this method, bacterial sample is streaked over the surface of a plated agar medium. During streaking, the cell density decreases, eventually leading to individual cells being deposited separately on the agar medium surface. Cells that have been sufficiently isolated will grow in to colony consisting only of the original cell type. Therefore, this is a very common method for isolation of bacteria.

Spread plate method

In this method the test sample is spread over the dry surface of an agar medium. The apparatus called spreader is used to perform this method.  Colonies that develop on its surface can count after proper incubation time. This method is employed to plate a liquid sample for the purpose of isolating or counting the bacteria present in that sample. Successful spread plate technique will result visible and isolated colonies of bacteria that are evenly distributed and countable in the plate. The most important advantage of this technique is no subsurface colonies appear in plate which leads the isolation of the organism easy.

Procedure of spread plate method

  • Prepare a dilution series at least 6-7 test tubes containing 9 ml of sterile distilled water.
  • Using a sterile pipette, add 1ml of test portion (sample) in the first tube of the set. Label it as 10-1.
  • Mix well by swirling the tube upside down few times.
  • From the first tube, take 1ml of the test portion and transfer to second tube. Label it as 10-2.
  • Repeat the procedure with all the remaining tubes labeling them until 10-6/10-7.
  • Pipette out 0.1 ml from each dilution onto the center of the surface of a pre-prepared and solidified agar plate.
  • Dip the L-shaped glass spreader into alcohol and flame the glass spreader over a Bunsen burner.
  • Spread the liquid sample evenly over the surface of agar using the pre-sterile glass spreader, carefully rotating the Petri dish anti-clockwise.
  • Incubate the plate at proper temperature as required.
  • Calculate the colony forming unit (CFU) value of the sample by following calculation method (see calculation)
Isolation of Bacteria | Bacterial Enumeration
Spread plate technique

Typical and atypical colonies

After the incubation period colonies are being counted. In many instances the total number of colonies is what is needed to be found. In certain other instances the number of colonies of a target organism is what is needed to be found. If the target organism produces separate distinguishable colonies, they can be counted without counting all the colonies of the plate. If all are counted it could be a total colony count. The characteristic colonies of the target organism are labeled as typical colonies while all other colonies known as atypical colonies.

Calculations

There is a method prescribe for calculations on solid media. These recommended ways of calculations are described in ISO 7218 which describes microbiological examination of food and ISO 8199 which describes general guidance on the enumeration of micro-organisms by water sample. The number of colonies appearing on solid plate has to be counted. The numbers can vary a lot. If the number of colonies exceed 300 those plates have to be rejected. Therefore, number of colonies on plates could be below 300. There are different ways of expressing the results when low numbers are there on the plates. Let us first learn how the numbers are calculated in the following situations.

  • Situation A – 10 to 200 total colonies on plates inoculated by spread plate method or using membrane filter method.
  • Situation B – Between 10 to total 300 colonies on plates inoculated by pour plate method (dilution plate method).
  • Situation C – Between 10 to 150 typical colonies in spread plate or pour plate.
  • Situation D – Between 10 to 100 typical colonies in spread plate method or pour plate.

In the counting of colonies each colony assume to have arising from one microorganism or from a single aggregate of microorganisms. Therefore, the result is expressed as the number of colony forming unit in a reference sample, reference volume of the sample. The reference sample volume is usually 100 mL or 1 mL.

Examples:

  1. For each dilution duplicates in pour plates were used for the calculation. Countable numbers appeared in two dilutions. The size of the inoculum was 1 milliliter. Find the number of CFU per milliliter.
spread plate calculations

Answer –

    CFU(ref)          = [(120+100+20+25)/ 1mL {(10-2×2) +( 10-3×2)}] 1 mL

                            = 1.2 × 10-4 CFU/mL

2. The following were the typical numbers of typical colonies appeared on the plates of given dilutions. How many target organism are in the sample?

Of the seventy colonies eight colonies were tested further for confirmation. Out of the eight only six colonies were found to be of the target organism. Similarly, ten colonies out of eighty colonies were tested and six were confirmed. From the plate that produce four colonies all four were tested and all for confirmed as belongs to the target organism. Out of eight colonies that appeared on the plates five were tested and four were confirmed.

spread plate calculations

Answer –

Number of target organism in seventy colonies = 70 × (6/8) = 52.5

Number of target organism in eighty colonies = 80 × (6/10) = 48

Number of target organism in eight colonies = 8 × (4/5) = 6.4

    CFU(ref)          = [(52.5+48+4+6.4)/ 1mL {(10-3×2) +( 10-4×2)}] 1 mL

                           = 5.0 × 10-4 CFU/mL

Special cases

  1. All plates of the test sample containing less than 10 colonies – Results expressed as “organism present in the volume studied”.
  2. No plates containing any colonies – Results expressed as “zero” or “organism non-detectable”.
  3. More than three hundred or two hundred total colonies with visible typical or confirmed colonies.

Examples:

spread plate calculations

Results expressed as the number of CFU per mL is 107 or above and less than 108.

4. More than three hundred or two hundred colonies without typical or confirmed colonies.

Example:

Results expressed as the number of total colonies must be less than 108 CFU per mL.

What is Enumeration?

Microorganisms carry out many important processes both in nature and in industries. E.g.: production of antibiotics, spoilage of foods, production of dairy products, and etc. In this case, counting (enumeration) of microorganisms in initial suspension is very important thing. The number of viable/total number of microorganisms in a product is useful quality indicator of contamination level/indicator of the success of a microbial process.

Isolation of Bacteria | Bacterial Enumeration
Classification of Enumeration methods

Dilution plate method

When bacteria are in very large numbers the dilution plate method is used to isolate and enumeration of microorganism (common for isolation and enumeration of bacteria). The technique use in here is pour plate technique. Therefore, sometime this dilution plate method is known as pour plate method. Environment like soil, contain large numbers of different bacteria. Therefore, the dilution plate method is recommended which reduces bacteria number by 10-fold in each dilution. Since the volume of the inoculum is known it is possible to enumerate bacteria using this method. Therefore, dilution plate method is a method that can be used for isolation as well as for enumeration. In enumeration what it gives is the total number of living bacteria (viable bacteria). It is usually called the total viable count and expressed as the number of “Colony Forming Units” per mL or per gram of a sample (CFU/mL, CFU/g).

In the dilution plate method what is counted is the number of colonies. It is assumed that each colony arises from a one cell. So, one colony means one cell. However, it is difficult to know whether the colony was formed by one cell or two or three cells (a few cells). Therefore, it is safe to count each colony as formed by one colony forming unit. What is obtained by the dilution plate technique is the number of colony forming units which are taken as equal to the number of viable cells.

Pour plate technique

Here, normally use 90 mm/100 mm diameter petri dish and 0.1 mL to 2 mL test portion.  In fact, test portion should be a one which gives less than three hundred colonies in the plate. The typical colonies out of the total colonies should be within 10 to 150. However, these numbers are not suitable (should be less) if the bacterium produces large colonies.

Principle of dilution plate method for isolation and enumeration of bacteria

Dilution plate method helps us to isolation and enumeration of bacteria as bacteria are often occur in very large numbers. It seems that the isolation & enumeration of them is going not to be easy. However, this method helps us to bring down the numbers per mL of billion times very easily making the isolation and enumeration easy. In this technique the plates which produces more than about 300 colonies have to be discarded for good reasons. Excessive numbers of colonies produced in the same plate will give a lot of over lapping colonies making the isolation incomplete and counting each CFU separately impossible.

What could be the exact reason not to take in to account the plates that produce low number of colonies such as below 20-30?

Different test books would say that it is not statistically correct. But that doesn’t explain the exact reason.  In this technique, people do not use the whole solution but only a 1 mL sample of the solution. This is practice first for the preparation of the dilution series & then to inoculate the plates from each dilution. When a solution carries a lot of bacteria the accuracy of sampling is higher. If a 100 mL solution carries a very large number of bacteria it is quite possible that a 1 mL sample taken out from the solution to be a representative sample of the solution. However, if the original solution carriers low concentration of bacteria, a small sample would not represent the solution. This happens due to fact that the bacteria are very thinly disperse in the solution and therefore there is no guarantee for each 1 mL volume to be similar to each other. Therefore, 1 mL sampling can be erroneous during the preparation of lower dilutions and during inoculating plates from lower dilutions. A very low dilutions is not suitable to take 1 mL sample. The 1 mL sample cannot be taken as representative of the entire solution. Therefore, the plates which give low numbers has to be discarded.

Important facts

Once the dilution series is prepared what is expected is to see a 10-fold reduction of CFU in each plate down the dilution series. Practical experiences showed that may be not same in sometimes. When the plate carriers 200 colonies the next plate down the dilution series must carry 20 colonies. If there is no clear 10-fold reduction alone the series that may be due to the reasons described above and also due to other errors like, pipetting errors. These errors can be minimized by taking the average CFU number in replicates.

Counting the numbers of harmful bacteria, the dilution plate method is often used to take the total number of bacteria, irrespective the times of bacteria. Therefore, discarding the plates produce low number of is accepted. If the testing bacteria are harmful even though few cells may be enough to cause the harm. Therefore, even a few colonies have to be taken into account.

There are accepted standards to follow when expressing these values. These standards agreed open all over the world for microbiology quality control.

Procedure of dilution plate method for isolation and enumeration of bacteria

  • Prepare a dilution series at least 6-7 test tubes containing 9 ml of sterile distilled water.
  • Using a sterile pipette, add 1ml of test portion (sample) in the first tube of the set. Label it as 10-1.
  • Mix well by swirling the tube upside down few times.
  • From the first tube, take 1ml of the test portion and transfer to second tube. Label it as 10-2.
  • Repeat the procedure with all the remaining tubes labeling them until 10-6/10-7.
  • Pipette out 0.1 ml from each dilution onto the pre sterile petri plate.
  • Pour 20 mL of autoclaved media into petri plate.
  • Spread the liquid sample evenly with agar by carefully rotating the Petri dish anti-clockwise.
  • Incubate the plate at proper temperature as required.
  • Calculate the colony forming unit (CFU) value of the sample (described under calculation part).
Isolation of Bacteria | Bacterial Enumeration
Dilution plate method/Pour plate technique

Most Probable Number Method (MPN) for isolation and enumeration of coliform bacteria

MPN is a method used often in various experimental fields of science. It is not only method used to enumerate coliforms or E. coli (oder Enterobacteriales) in food & water microbiology. MPN method helps specially in the of enumeration of low numbers. In the case of food and water microbiology it gives positive result even if single cell is present. Such low numbers cannot produce accurate results in many other techniques including dilution plate method.  In food and water microbiology even, a single cell can give clear positive result in the fermentation tube.

Isolation of Bacteria | Bacterial Enumeration
Isolation of coliform bacteria by using MPN technique

The above diagram describes the very basic principle of MPN counting that helps us to understand the coliform testing in water. In that method, test portions are 10 mL, 1 mL and 0.1 mL in five parallel tubes. In other words, three successive dilutions in five parallel tubes. As coliforms or E. coli, if present at all, occur in low numbers in water, using the particular test portions as three successive dilutions in five parallel tubes. As the numbers are low the particular dilutions are going to give at least some negative tubes. Therefore, five parallel tubes in the given dilutions provide a suitable test setup for coliform testing in water.

If the numbers are higher further dilutions would be needed. If the test organism does not provide a clear result in liquid broth, testing on solid media will be required. Coliforms give a clearly distinguishable result in MacConkey broth/ Lauryl sulphate broth by producing acid and gas. If test organism is an organism that cannot produce such distinguishable result in liquid medium and instead produce only sign of growth (turbidity), testing in liquid medium may not be suitable to enumerate such bacteria. In water testing there may be instance where we have to test food for such bacteria. In such occasions solid medium are to be utilize. Even with coliform testing in liquid media a confirmation has to be done using EMB (Eosine Methylene Blue Agar) plates and IMViC test.

IMViC test

Genus Indole Test MR Test VP TEST CITRATE TEST

Escherichia

+
+
-
-

Enterobacter, Citrobactor, Klebsiella

-
-
+
+

In water quality testing five tubes are prepared for each dilution. In food testing 3 test tube replicates readings are prepared for each dilution. The number of positive and negative tubes in the consecutive dilutions take into account. The most probable number of coliforms present in the original samples can be found in MPN table.

Procedure of MPN Method

This method is consisting of three main steps including presumptive test, confirmed test and completed test.

Presumptive test

  • Prepare medium (either MacConkey broth or Lauryl sulphate broth) in single and double strength concentration.
  • Take five tubes of double strength and ten tubes of single strength for each water sample to be tested.
  • Add a durham tube in inverted position.
  • Sterilize by autoclaving at 121°C temperature (15 lbs pressure) for 15 minutes.
  • Disperse 10 mL (100) of each dilution into each of five tubes containing 10 mL of double strength Lauryl sulphate broth or MacConkey broth (If you are going to test food sample, use three test tubes except five tubes).
  • Disperse 1 mL (10-1) of each dilution into each of five tubes containing 10 mL of double strength Lauryl sulphate broth or MacConkey broth.
  • Disperse 0.1 mL (10-2) of each dilution into each of five tubes containing 10 mL of double strength Lauryl sulphate broth or MacConkey broth.
  • Incubate the tubes at 37 °C for up to 24 hours. If no tubes appear positive re-incubate up to 48 hours.
  • Record the number of positive tubes. Any tubes producing gas in broth is considered as positive for the presence of coliforms)
  • Calculate the presumptive coliforms by using of MPN table.

Confirmed test

  • Sub culture a loopful from each of the positive tubes in presumptive test in to one set of 10 mL quantities of BGLB broth (with inverted durham tubes) pre-warmed to 44 °C.
  • Incubate all tubes at 37 °C for up to 48 hours.
  • Records all tubes positive for gas after 48 hours of incubation as positive for confirmed coliforms.
  • Calculate the confirmed total coliforms by using of MPN table.
  • Sub culture a loopful from each of the positive tubes in presumptive test in to another set of 10 mL quantities of BGLB broth (with inverted durham tubes) pre-warmed to 44 °
  • Incubate all tubes at 44 °C for up to 48 hours.
  • Records all tubes positive for gas after 48 hours of incubation as positive for fecal coliforms.
  • Calculate the confirmed total fecal coliforms by using of MPN table.

Completed test

  • Sub culture all positive tubes from confirmed test (for 44 °C incubated set) by streaking on EMB agar plate.
  • Incubate plates at 37 °C for up to 24-48 hours.
  • Examine for typical coli colonies pick one colony from each plate into separate tubes of peptone water.
  • Incubate at 44 °C for up to 48 hours.
  • Test peptone water for the production of indole by adding 0.2 mL of Kovac’s reagent after 48 hours. A dark red color in the surface layer constitutes a positive test.
  • Culture showing indole production are considered to be positive for coli.

Calculations and expression of results

  • Record the number of tubes of each set of five, which are positive for the particular organisms to be estimated.
  • Select the highest dilution in which all three tubes are positive and the next two higher dilutions. If this is not possible because none of the dilution show five positive tubes, select the last three dilutions.
  • Refer to the MPN table, multiply the MPN index obtained from the table by the reciprocal of the lowest dilution selected to obtain the MPN per gram or milliliters of sample.

Coliform bacteria

Coliform bacteria are a commonly used bacterial indicator of quality of foods & water. They are defined as rod-shaped Gram-negative, non-spore forming and motile/non-motile bacteria which can ferment lactose at 35 °C within 48 hours producing acid and gas. Escherichia coli (E. coli) which is an intestinal organism is one of the most available coliforms in nature. There are some other coliforms which are non-fecal. Enterobacter, Klebsiella and Citrobacter are coliform organism live in soil. If coliforms are present in water or food, it is possible to have fecal pollution. However, it has to be found out whether the coliform is a fecal coliform or a non-fecal coliform such as Enterobacter. However, Enterobacter, Klebsiella and Citrobacter are not exclusively fecal origin.

If coliforms are there in 100 mL of water, the distribution of water for drinking and cooking must stop. However, further investigations need to find out whether this coliform is fecal or non-fecal. Fecal and non-fecal coliforms can be distinguished by the ability of fecal coliforms to do lactose fermentation at 44°C. Only fecal coliforms can grow on lactose at 44 °C. If the test is positive for fecal coliforms further tests should done to confirm that it is E. coli.

Membrane filter method

The volume of filtration depends on the number of bacteria and filterability of the liquid. The volume should be at least 10 mL. This technique is suitable for water that contain not too much particulate for colloidal matter in suspension. It is suitable to test drinking water. The pore size is 0.45 µm or less. Some small organism like legionella need a pore closer in order to get above 10 to 100 typical colonies on a 50 mm filter.

 The total number of colonies on filter should be less than two hundred. For larger colonies the expected number should be lower than that. After filtration the membrane can be placed on Agar. A thin layer of agar can be laid above the membrane if needed. After the filtration step the membrane should be transferred to selected media for purposes of selection or differentiation of organisms thus allowing isolation and enumeration of discrete colonies of bacteria. After 24 hours incubation period results can be obtained more rapidly than by the MPN standard methods. It provides only presence or absence result after 24 hours.

Counting chambers

These are slides with cavities of known microscopic volumes on the surface. Place a drop of broth culture on the slide and cover with a cover slip. Now the cavity has entrapped a small known volume. The numbers of bacteria in cavity can be counted because the volume is known. Therefore, total number of cells per mL can be counted. There are two common counting chambers are using in nowadays in the world.

  1. Petroff-Hausser counting chamber
  2. Haemocytometer

Petroff-Hausser method is popular for bacteria and sperm counting and offered in series of cell depths. Hemocytometer is a specimen slide which is used to determine the concentration of cells in a liquid sample (to count blood cells/WBC, RBC, FBC).  

Isolation of Bacteria | Bacterial Enumeration by using Haemocytometer
Enumeration of bacteria by using Haemocytometer

Breed’s smear method

This this method uses in counting bacteria in milk. It is a rapid method. A 1 cm2 square is marked on the slide. A known small volume spreads on that area which observes under the microscope. It is possible to count the number of bacteria seen in the microscopic field. The microscopic field is the large illuminated circle seen under the microscope. In fact, this is only a very small area of the total Breed’s smear. However, if the actual area of the circle (microscopic field) can be found out the total number of bacteria in the entire Breed’s smear can be calculated. To measure the area of the microscopic field its radius should be known. This can be done by putting a micrometer on the stage of the microscope (Micrometer is a transparent meter scale). The stage micrometer has to be placed across the center of the microscopic field in order to measure the radius. The center of circle can be located with the help of a small disc (which has a line running across the center) in the eye piece of the microscope.

When this is in the eye piece a line (divided into equal parts) is seen running across the center of the microscopic field. The stage micrometer must be placed aligned with the line running across the center of the microscopic field. The diameter of the microscopic field can be measured in this manner and the area can be calculated using πr2.

The accuracy of the method can be increased by counting the number of bacteria in several microscopic fields and taking the average number per microscopic field.

Electronic cell counters

Bacteria in a broth culture (liquid culture) send through small orifice (hole) which can allow one cell of a time to go through it. The conductivity across the hole changes when cell passes through it and the electronic counts it as one. What it gives is the total count (viable cells and dead cells). The common apparatus for this is Coulter counter.

Article By,

Malki Narmada – Microbiologist

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