A variety of techniques has been developed for the isolation of microorganism, mainly the bacteria, from the specimen or from the sample cultures and Spread plate culture technique is among the most widely used culture technique for isolating the bacteria.
The Culture techniques are commonly used in the laboratory for various purposes for which they are intended. The indications for the culture of the organism is mainly done –
- To demonstrate the cultural characteristics of the bacteria (e.g. color, texture, size, elevation etc.).
- To isolate the bacteria in discrete colonies from the specimen containing more than 1 bacterium.
- For determining the Sensitivity and/or Resistance of bacterium towards the particular Drug/Antibiotics or Test substances.
- To obtain the sufficient growth of the bacterium for various biochemical and other tests.
- To estimate the viable counts of the bacteria in the specimen.
- To maintain the stock cultures.
- To transport or short-term storage of the specimen (e.g. stab culture).
Three types of techniques are commonly employed for the isolations of microorganism from the specimen which are as follows:
- Pour Plate Technique
- Spread Plate Technique
- Streak Plate Technique
In this article, we’ll discuss the Spread Plate technique for the isolation of microorganism in the microbiology laboratory.
PRINCIPLE OF SPREAD PLATE CULTURE TECHNIQUE
The spread plate culture method is one of the commonly used culture technique for the isolation of microorganisms, especially the bacteria, in the laboratory. In this technique, a serially diluted specimen containing 2 or more bacteria or microbe (Mixed culture) is used which is spread over the solidified agar media plates as a thin layer with the help of a sterile L-shape glass rod (Spreader) while the media plate is spun on a turntable.
The principle behind this method is that when the Media plate is spun, at some stage, single cells will be deposited with the bent glass rod (Spreader) onto the surface of the Agar media. Some of the cells present in the specimen / diluted specimen will be separated from each other by a distance sufficient to allow the colonies that develop to be free from each other.
REQUIREMENTS FOR THE SPREAD PLATE TECHNIQUE
- 24 hours old nutrient broth culture of two or more bacteria (Mixed Culture) or Sample/Specimen.
- Nutrient Agar Medium or Any other Media
- Six 9 ml Sterile Water Blanks
- Sterile Petri plates
- Graduated pipette (1ml or 1000 ml)
- Graduated pipette (0.1ml or 100 ml)
- L-shape Glass Spreader
- 95% Ethanol or Isopropyl alcohol
- Bunsen burner or Spirit lamp
PROCEDURE OF SPREAD PLATE CULTURE TECHNIQUE
⇒ Prepare the Nutrient agar medium (NAM) and label the Sterile Petri plates as number 1 to 6. Place the labeled tubes in the test tube rack.
⇒ Now, pour the prepared Nutrient Agar Medium into the Sterile Petri plates under strict aseptic conditions. Allow the Media plates to Solidify.
Serial Dilutions of the Specimen / Sample
⇒ Label the 6 Sterile Water blanks (9ml sterile water in each tube) as number 1 to 6 with the help of Marker.
⇒ Mix well the 24 hours old broth culture or the specimen to equally distribute the bacterial cells in the tube.
⇒ After mixing, Remove the Cotton plug and aseptically transfer the 1 ml of the microbial suspension from the tube of culture to sterile water blank tube no. 1 using a graduated pipette.
⇒ Shake the tube no. 1 to mix well the content to uniformly distribute the bacterial cells. Transfer the 1 ml of this to the water blank tube no. 2 by using the graduated pipette.
Note: Use the separate sterile pipette each time to transfer the contents from one tube to another.
⇒ In this way, make serial dilutions till the six water blanks (no. 1 to no. 6).
Inoculating the Specimen / Sample on Media plates
⇒ Take 95% Alcohol in a beaker and Dip the L-Shape Glass Spreader in it.
⇒ Now, transfer the 0.1 ml or 100 ml of the bacterial suspension each from the tube no. 1 to 6 to the Solidified Agar Media Plates labeled as 1 to 6 by using separate sterile pipette each time.
⇒ Remove the Glass rod from the Alcohol and sterilize the bent portion in the Bunsen burner or Spirit lamp flame. Cool it down for 15—20 seconds.
⇒ Place the Media plates containing specimen on to the Petri plate turntable, Remove the Cover/Lid or the Media plate and spin the turntable.
⇒ Lightly touch the sterile spreader to the agar media surface and move it back and forth while the turntable is spinning to evenly spread the specimen over the agar media surface.
⇒ Replace the cover / Lid of inoculated Media plate when the Petri plate turntable stops spinning.
⇒ Immerse the L-shape glass spreader in the Alcohol beaker and flame it to sterilize.
⇒ Repeat the step with all the Media plates and sterilize the Glass spreader each time after using.
⇒ Incubate the plates in an inverted position at optimum temperature (usually 37 °C) for 24 – 48 hours.
Examine the plates for the appearance of individual colonies growing throughout the agar medium.
RESULTS OF SPREAD PLATE TECHNIQUE
The colonies in the cultures media plates inoculated by the serial dilutions of the specimen will shows the lesser and lesser no. of the colonies with the increase in dilution factor that means the highest no. of colonies will develop in Plate no. 1 and least no. of colonies in plate no. 6 which will be distributes more or less sparsely in the entire plate.
Select the discrete and well-developed colony and note down the characteristics like Color, Shape, Size, Appearance, Elevation, and Pigmentation etc.
These colonies may be transferred i.e. sub-cultured to the fresh media plates by streaking to obtain the pure culture of the bacterial cells for further study.
PRECAUTIONS TO BE TAKEN…..
⇒ The protocol should be followed under all aseptic conditions preferably in Laminar Air Flow (Safety cabinet) to avoid any contamination.
⇒ Accurately measure the quantity while preparing the serial dilutions of the specimen.
⇒ Allow the media plated to uniformly solidify. Do not inoculate the specimen on partially solidified media plates.
⇒ Accurately measure the quantity of Diluted specimen while inoculating onto the Solidified Media Plates.
⇒ Uniformly spread the specimen onto the Media plate to get the discrete & well-developed colonies.
⇒ Sterilize the L-shape Glass Spreader each time after using it to avoid any errors and contaminations.