e-portfolio sixth week

      During the lab this week, I had done 3 experiments which is experiment 6( preparation of bacterial smear), experiment 7( simple staining) and experiment 8 (negative staining).All the experiment we did used gram-negative bacteria. For the bacterial smear we have done bacterial smear  to be used for the simple staining and negative staining experiment. The bacteria we used are Staphylococcus aureus, Bacillus cereus, Escherichia coli, Micrococcus Luteus and Bacillus subtilis. There are two type of bacterial smears which are the smears from a broth medium and the smears from a solid medium. The bacterial smears we done are the smears from solid medium which is the agar plate. The process of carrying out bacterial smear follow the following procedures. First, clean the glass slides by flaming it. Then, using a loop, place one to two loops of water on each slides. Then, with a sterile loop, we touch the entire loop to the culture and emulsify the cells in water on all the slides respectively. Next, we allow the slides to air-dry completely by placing them on the bench.Finally, we heat fix the preparation.

   We did simple staining for Escherichia coli, Bacillus cereus and Staphylococcus aureus.The stains we used are the carbolfuchsin, methylene blue and crystal violet. We did it in a group and each bench must prepare two sets of the simple staining of the bacteria for each different stains. We prepared 18 slides in total. For the procedures of the simple stain, we used the bacterial smears we prepared during experiment 6. First, place a slide on the staining tray and flood the smear with one of the indicated stains, using the appropriate exposure time for each: carbolfuchsin,15-30 second; crystal violet,20-60 seconds; methylene blue, 1-2 minutes. Then, we gently wash the smear with tap water to remove excess stain. During this step, hold the slide parallel to the stream of water, in this way, I can reduce the loss of organisms from the preparation. Next, we blot dried the slide by using bibulous paper. We did the procedures for all the organisms by using all three different type of stains for each organisms.

 For negative stains, the background is stained dark while the bacteria remains colourless. This is because the stain we used are nigrosin which acidic stain which negatively charged chromogen repel with the gram-negative bacteria. So, the bacteria resists staining and remained colourless. The bacteria we used are Micrococcus luteus, Bacillus cereus and Staphylococcus aureus. The procedures of negative staining is as followed. First, we placed a small  drop of nigrosin close to one end of a clean slide. Then, by using aseptic technique, we placed a loopful of inoculum from the M. luteus culture in the drop of nigrosin and mix. We then placed a slide against the drop of suspended organisms at 45 degree angle and allow the drop to spread along the edge of the applied slide. Next, we push the slide away from the frop of suspended organisms to form a thin smear. We air-dried the slides. Heat fix should not be done for negative staining. We repeat the steps 1-4 for slide preparations of the remaining cultures, We then examined all the slides under high power and oil immersion and recorded the result in the Lab report.

Simple staining using crystal violet, methylene blue and carbolfuchsin and negative staining using nigrosin for E. coli

The slide of E. coli of carbolfuchsin (simples staining)

The slide of E. coli for negative staining

The slide of S.aureus for simple staining (crystal violet)

 

 The slide of B.cereus using methylene blue (simple staining)