A bacterial smear is required particularly for the microscopic examination of the specimen or the culture for the identification of bacteria which is done by preparing the smear from the specimen or culture followed by staining with the appropriate stains, e.g. Gram staining. The purpose of making the smear is to fix the specimen or the bacterial cells from the culture onto the microscopic glass slide so that it will not get washed away during the staining procedure. A bacterial smear can be prepared from the specimen itself or from the Liquid (Broth) cultures & Solid (Agar) cultures of the specimens
The general procedure of preparing an ideal bacterial smear is described below……
MATERIAL REQUIRED FOR PREPARING A BACTERIAL SMEAR
- Specimen or Specimen culture, on Nutrient Agar media & in Nutrient Broth
- Clean Glass Slides
- Inoculating Loop
- Bunsen Burner/Spirit Lamp
PROCEDURE FOR PREPARING AN IDEAL SMEAR
⇒ Take a Clean, Grease free microscopic glass slide and mark the smear area on the underside of the slide with a glass marking pencil.
⇒ Flame the slide and place it on the disinfected table. For broth culture flame the slide on the same side on which the smear will be prepared.
⇒ Flame the loop at 60° angle into the Bunsen burner/ Spirit lamp flame by holding the inoculating loop in the right hand.
⇒ Heat till the entire wire portion of the inoculating loop appears to be red hot and allow it to cool.
FOR BROTH CULTURE
⇒ Shake the content of the tubes for the uniform distribution of microorganisms throughout the broth.
⇒ Pick up the culture broth tube with your left hand.
⇒ Remove the Cotton plug with the little finger of the right hand.
⇒ Immediately, Flame the mouth of the test tube containing broth culture.
⇒ Insert the Sterilized loop into the culture tube and take out a loopful of cultured broth.
⇒ Immediately, flame the mouth of the test tube containing cultured broth and replace the plug.
⇒ Now, Place the loopful of the cultured broth onto the clean & sterilized microscopic glass slide and flame the inoculating loop.
⇒ Spread the bacterial suspension thinly to over an area about the size of a silver coin (of USA) or 10 paisa coin (of INDIA) with the sterilized inoculating loop.
FOR AGAR CULTURES
⇒ Put a loopful of sterilized water on clean, grease-free and sterilized microscopic glass slide.
⇒ Aseptically transfers a small amount of bacterial growth (colony) from the cultures Nutrient Agar Media (NAM) plate to the sterilized water droplet with the help of sterilized inoculating loop.
⇒ Emulsify the Cultured bacteria into the water droplet with the inoculating loop and spread the bacterial suspension thinly to over an area about the size of a silver coin (of USA) or 10 paisa coin (of INDIA) with the sterilized inoculating loop.
⇒ Air dry the smear at room temperature.
FIXING THE SMEAR
⇒ Gently heat the slide by holding it in the right hand well above the blue portion of the Bunsen flame 3-5 times.
⇒ Allow the smear to cool at room temperature then follows the staining protocol.
An ideal bacterial smear should be thin, semi-transparent, whitish layer or film, circular with diameter 1 cm. (approximately) free from dirt, dust or any contamination.