Preparation of bacterial smear (Laboratory exercise)

Use fresh cultures between 24-48 hours old, whenever possible. When making smears, use a medium-sized drop of water and a small amount of bacteria. Mix the bacteria in the drop quite well with an inoculating needle, and spread it out thinly. A smear that is too thick will not only be difficult to stain properly but it will also be very difficult to observe individual bacterial cells under the microscope.
Use fresh cultures between 24-48 hours old, whenever possible.



Materials

Glass slide | Bunsen burner | tube of sterile water | slide warmer | gloves | pencil |inoculating needle |sterile transfer pipette (“transpette”) | inoculating loop | culture of Staphylococcus epidermidis and Escherichia coli

a. Take your streak plates and examine them for the two different colony types. The TSA plate should have well-isolated Staphylococcus epidermidis (Gram-positive) and Escherichia coli (Gram-negative) colonies. The PEA and MSA should only have one colony type (S.epidermidis), and the MAC and EMB should only have one colony type (E.coli).

b. Assemble the materials necessary for making the smears.

c. With a pencil, label two glass slides on the frosted end with the names of the respective test organisms: Staphylococcus epidermidis and Escherichia coli.

d. Using the aseptic techniques put a medium-sized drop of water on the slide in the center, using a sterile pipette or an inoculating loop. Transfer a small amount from a single, well-isolated colony from the Petri plate to the drop of sterile water on the slide. When transferring an isolated colony from the streak plate, an inoculating needle rather than a loop is used.

e. Touch the inoculating needle to the center of a well-isolated colony. You may use any one of your plates. However, if you use a selective agar, remember that the bacterial type that did not appear to grow is only inhibited. Therefore, you should touch the needle to the very top or edge of the colony without going too deep. DO NOT TOUCH THE AGAR SURFACE! 

Transfer the colony aseptically to the appropriately labeled glass slide and thoroughly mix the bacteria with the drop of sterile water on the slide.

f. Repeat the procedure for the other colony type.

g. Let each smear air-dry thoroughly and then heat-fix gently using either the flame of a Bunsen burner or a slide warmer. Heat-fix the bacteria onto the slide by passing the slide, smear side up, quickly through the flame of the bunsen burner 4-5 times. Avoid getting the slide too hot; this will cause distortion of the morphology of the cells. 

This step will keep your smear from washing off of the slide during the staining procedure. These smears will be used to perform the Gram Stain procedure.



Reference
http://coproweb.free.fr/pagbac/introbac/smearpre.htm
https://www.scienceprofonline.com/microbiology/how-to-prepare-microscope-slide-of-bacteria.html
https://bio.libretexts.org/Bookshelves/Ancillary_Materials/Laboratory_Experiments/Microbiology_Labs/Book%3A_Laboratory_Exercises_in_Microbiology_(McLaughlin__Petersen)/3%3A_Preparation_of_Bacterial_Smears_and_Introduction_to_Staining
http://spot.pcc.edu/~jvolpe/b/bi234/lab/labTechniques/bacterial_smears.htm
https://openwetware.org/wiki/BISC209:_Preparing_a_bacterial_smear_slide
https://uomustansiriyah.edu.iq/media/lectures/6/6_2017_03_11!10_58_07_PM.pdf
https://www.labce.com/spg603164_preparation_of_a_gram_stained_smear_from_culture.aspx
https://milnepublishing.geneseo.edu/suny-microbiology-lab/chapter/differential-staining-techniques/
http://vlab.amrita.edu/?sub=3&brch=73&sim=208&cnt=2

Post a comment

0 Comments