How to Prepare An Ideal Bacterial Smear ?

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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

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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.

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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.

Frequently Asked Questions (FAQs)

Q1. What is a bacterial smear?

A bacterial smear is a thin layer of bacteria that is spread onto a glass slide for observation under a microscope.

Q2. Why is a bacterial smear important?

A bacterial smear is important because it allows microbiologists to observe and study the morphology, staining properties, and arrangement of bacterial cells.

Q3. What are the materials needed for preparing a bacterial smear?

The materials needed for preparing a bacterial smear include a glass slide, a bacterial culture, a flame source (such as a Bunsen burner), and a staining agent.

Q4. How do you prepare a bacterial smear?

To prepare a bacterial smear, first place a drop of water on a clean glass slide. Next, using a sterile loop or a sterile swab, transfer a small amount of the bacterial culture onto the water drop. Then, using a second clean glass slide, spread the bacterial culture evenly over the surface of the slide. Finally, air-dry the slide.

Q5. What is the purpose of heat-fixing a bacterial smear?

Heat-fixing a bacterial smear helps to adhere the bacterial cells to the glass slide, prevent the cells from washing away during the staining process, and kill the bacteria.

Q6. How do you heat-fix a bacterial smear?

To heat-fix a bacterial smear, hold the slide with the bacterial smear over a Bunsen burner flame for a few seconds until the slide feels warm to the touch.

Q7. What is the purpose of staining a bacterial smear?

Staining a bacterial smear helps to visualize the morphology and arrangement of bacterial cells.

Q8. What are the types of staining methods used for bacterial smears?

The types of staining methods used for bacterial smears include Gram staining, acid-fast staining, and endospore staining.

Q9. What is Gram staining?

Gram staining is a differential staining method used to distinguish between two different groups of bacteria based on the properties of their cell walls.

Q10. What is acid-fast staining?

Acid-fast staining is a differential staining method used to identify bacteria with a unique cell wall structure, such as Mycobacterium tuberculosis.

Q11. What is endospore staining?

Endospore staining is a differential staining method used to visualize the presence and location of bacterial endospores.

Q12. What is the importance of properly fixing and staining a bacterial smear?

Properly fixing and staining a bacterial smear is important because it allows for accurate and reliable observation and identification of bacterial cells.

Q13. What are the common mistakes made when preparing a bacterial smear?

Common mistakes made when preparing a bacterial smear include using too much bacterial culture, not allowing the slide to air-dry completely before heat-fixing, and over-staining or under-staining the smear.

Q14. How can you avoid contamination when preparing a bacterial smear?

To avoid contamination when preparing a bacterial smear, use sterile instruments and work in a clean and sterile environment.

Q15. How long can a properly prepared bacterial smear be stored?

A properly prepared bacterial smear can be stored for several weeks if kept in a dry and cool environment.