Page 56 - MNUmicrobiology practical 2025

General Microbiology & Immunology (PM 401) second level Semester 4 2024/2025

-Gram’s iodine [mordant - makes primary stain fix to cell wall]

-Decolorizer (95% ethyl alcohol) [washes stain out of cell walls with high lipid

content]

-Gram’s safranin [counterstain]

5- Inoculating loop

6-Bunsen burner

7- Slide holder.

8- Washing water

9- Microscope with oil immersion lens

10- Marker pen

Gram staining steps

1- Prepare of bacterial smear

2- Flood crystal violet solution over fixed smear

3- After 30 – 60 seconds, pour off the CV solution and rinse with gentle running water.

4- Flood the Gram’s Iodine solution over the smear

5- Leave the iodine solution for 30 – 60 seconds and pour off the excess iodine and

rinse with gentle running water

6- Shake off the excess water over the smear

7- Decolorize the smear by adding a few drops of decolorizing solution and shake

gently and rinse with distilled water after 5 seconds.

8- Rinse with distilled water to wash decolorizer

9- Shake off the excess water over the smear

10- Pour counter stain over the smear

11- Leave for 30 – 60 seconds and wash with gentle running water

General Microbiology & Immunology (PM 401) Practical note Page 45