Methods of Study in Medical Bacteriology

Question 1

Outline the steps of Gram staining.

Answer 1

(1) Smear preparation: A thin film of bacteria is smeared on a glass slide and heat-fixed.
(2) Primary staining: The smear is stained with crystal violet, which stains all bacteria purple.
(3) Mordant application: Gram’s iodine is added, which forms a complex with the crystal violet, trapping it in the cell wall.
(4) Decolorization: The smear is decolorized with alcohol or acetone. Gram-negative bacteria lose the crystal violet stain, while gram-positive bacteria retain it.
(5) Counterstaining: A counterstain, usually safranin, is applied to stain the decolorized gram-negative bacteria pink or red. This makes them easily visible under a microscope.

Further notes:
✔ In gram-negative bacteria, the decolorizer dissolves the outer membrane, making the cell wall leaky. This allows the crystal violet-iodine complex to escape, and the cell loses its purple color.
✔ In gram-positive bacteria, the decolorizer dehydrates the thick peptidoglycan layer, trapping the crystal violet-iodine complex even more tighly.

Question 2

Name four types of staining techniques in bacteriology.

Answer 2

(1) Simple staining: utilizes one type of stain
(2) Negative staining: stains the background and enables certain features to be visualized clearly against stained background
(3) Differential staining: involves application of more than one reagent (e.g. Gram’s stain)
(4) Fluorescence staining/Immunofluorescence staining: involves staining with fluorescent materials; stained materials are examined by fluorescence microscope and ultra-violet light

Question 3

Name 5 types of microscopes that may be used to study bacteria.

Answer 3

(1) Light microscope
(2) Phase contrast microscope
(3) Fluorescence microscope
(4) Dark field microscope
(5) Electron microscope

Question 4

Briefly discuss the types of artificial culture media.

Answer 4

(1) Simple media: for general use; contain peptone (polypeptides and amino acids); support the growth of many types of bacteria; include nutrient broth, nutrient agar
(2) Selective media: useful in isolation of a specific organism from a specimen which might have other bacteria which might interfere with the growth of the specific organism; contain chemicals or antimicrobial agents; inhibit the growth of any other bacteria that might be in a specimen e.g. Thayer-Martin medium, Deoxycholate Citrate Agar
(3) Enriched media: contain nutrients for isolation of fastidious bacteria which require special nutrients; blood agar, chocolate blood agar
(4) Enrichment media: liquid media or broths with selective properties; alkaline peptone water, selenite F broth, tetrathionate broth
(5) Transport media: contain various substances which protect delicate bacteria in specimens from harmful substances which would make them nonviable; useful for transportation of specimens when there is a delay from the time of collection to the time of specimen delivery and processing in the laboratory; Stuart’s transport medium, Cary-Blair transport medium
(6) Indicator media: components include fermentable carbohydrates and chemical indicators; associated with color change due to changes in pH caused by acid production as a result of carbohydrate fermentation by the bacteria; MacConkey’s medium, CLED medium