Experiment 6: Preparation of bacterial smear, Experiment 7: Simple staining and Experiment 8: Negative staining

      Assalamualaikum and every good evening to everyone that read my post... The experiment I did in this week which is sixth week of basic technique of microbiology have three experiment. First is preparation of bacterial smears, next is simple staining and the last one is negative staining. Bacterial smears must be prepared prior to the execution of any of the staining technique. Bacterial cells are usually colorless because cytoplasm, for the most part, is transparent. Since the bacteria are colorless, it is almost essential to add a stain to make the bacteria more visible. Once stained, cell morphology can be observed. In simple staining, the bacterial smear is stained with a single reagent, which produces a distinctive contrast between the organism and its background. In negative staining requires the use of an acidic stain such as India ink or nigrosin. The acidic stain, with its negatively charged chromogen, will not penetrate the cells because of the negative charge on the surface of bacteria. First, I thought the experiment was more difficult than the previous experiment but it is like vice versa. These three experiments are easiest than previous one. As usual, Encik Hussain taught and gave some demo how to carry out the experiment to us. For a change Dr Adelene also gave some demo to another three groups.

      In simple staining, although not difficult, the preparations requires adequate care. Meticulously follow the rules listed which are preparation of the glass microscope slide, labeling of slides, preparation of smear in broth cultures and cultures from solid medium and heat fixation. In preparation of the glass microscope slide, if any grease or oil from the fingers on slides with soap and water or scouring powders such as Bon Ami, followed by a water rinse and a rinse of 95% alcohol. In labeling of slides, the initials of the organism can be written on either end of the slide with a glassware marking pencil on the surface on which the smear is to be made. In prepration of smear, a thick or dense smear occurs when too much of the culture is used in its preparation, which concentrates a large number of cells on the slide. Broth cultures, depending of the size of the loop, one or two loopfuls should be applied to the center of the slide with a sterile inoculating loop and spread evenly over an area about the size of a dime. Cultures from solid medium, organisms cultured in a solid medium produce thick, dense surface growth and are not amenable to direct transfer to the glass slide. In heat fixation, unless fixed on the glass slide, the bacterial smear will wash away during the staining procedure.

 
    Simple staining can be used to determine cell shape, size and enlargement. The simple stain procedure involving only one stain. We can choose from methylene blue, Gram safranin and Gram crystal violet. Basic stain such as methylene blue, Gram safranin and Gram crystal violet are useful for staining most bacteria. These stain will readily give up a hydroxide ion or accept a hydrogen ion, which leaves the stain positively charged. Since, the surface of most bacterial cells is negatively charged, these positively charged stain adhere readily to the cell surface.

   Negative staining is an established method, often used in diagnostic microscopy, for contrasting a thin specimen with an optically opaque fluid. In this technique, the background is stained, leaving the actual specimen untouched, and thus visible. This contrasts with 'positive staining', in which the actual specimen is stained. Negative staining is typically performed using a black ink fluid such as nigrosin. The specimen, such as a wet bacterial culture spread on a glass slide, is mixed with the negative stain and allowed to dry. When viewed with the microscope the bacterial cells, and perhaps their spores, appear light against the dark surrounding background. An alternative method has been developed using an ordinary waterproof marking pen to deliver the negative stain.

                                                Staphylococcus aureus using methylene blue

Escherichia coli using crystal violet

Bacillus cereus using carbol fuschin

Negative stain of Micrococcus luteus

Negative stain of Bacillus cereus

Negative stain of Staphylococcus aureus