LabEx1: staining procedures

Question 1

Gram staining procedures?

Answer 1

1. Crystal violet for 1 min (Primary)
2. Wash with water
3. iodine for 1 min (Mordant)
4. Alcohol 10sec (decolorize)
5. wash with water
6. Safranin for 30sec (counter stain)
7. Wash and blot dry

Question 2

Spore Stain Procedures?

Answer 2

1. Cover the slide with a blotting paper strip
2. flood with malachite green (primary) 60 sec
3. put over flame for 4min and do not let it dry, just add more malachite green to keep it soak (mordant)
4. remove paper (let slide cool for 1min)
5. wash slide with water (decolorizing agent)
6. Safranin for 1 min (counter stain)
7. wash with water and blot dry

Question 3

Acid Fast Staining Procedures?

Answer 3

1. cover the slide with a blotting paper strip
2. flood with carbolfuschin for 60 sec
3. put over flame for 4min and do not let it dry, just add more carbolfuschin to keep it soak (mordant)
4. remove paper (let slide cool for 1min)
5. Alcohol for 10 sec or until slides is clear (decolorize)
6. wash the slide
7. methylene blue for 1min (counter stain)
8. wash with water and blot dry

Question 4

Streak plate procedures?

Answer 4

1. Draw 4 quadrant on the streak plate
2. sterilize the loop
3. get 1 loop full from the mixed culture
4. streak zigzag from quadrant 1 to 4
5. sterilize loop for each quadrant but dont loop again for 2 to 4
6. DO NOT CUT ACROSS NO MORE THAN 2 TIMES

Question 5

EMB plate procedures

Answer 5

1. Draw 4 quadrant on the EMB plate
2. sterilize the loop
3. get 1 loop full from the mixed culture
4. streak zigzag from quadrant 1 to 4
5. NO NEED TO sterilize loop for each quadrant
6. DO NOT CUT ACROSS NO MORE THAN 2 TIMES

Question 6

Smearing procedures?

Answer 6

1. sterilized the slides and loop
2. transfer few loopfuls of sterile water
3. sterilized the loop again
4. carefully loop the bacteria from the tube or plate
5. smear the loop of bacteria on the slide with the sterilize water.
6. air dry by pass the slide through the flame 2 to 3 times