LAB PRACTICAL 2: Introduction to Bacteria: Morphology and Staining

Bacteria are single celled microbes. The cell structure is simpler than that of other organisms as there is no nucleus or membrane bound organelles. 

Morphology

One of the first steps in classifying bacteria is by observing their morphology. The morphology can be divided into several categories: 

1. Shape of colony
2. Shape and arrangement of cells
3. Subcellular organization

Observation: 

Bacillus

Elevation: Flat

Margin: Undulate

Form: Circular 

E. coli

Elevation: Convex

Margin: Undulate

Form: Circular, mucous

Pseudomonas

Elevation: Convex

Margin: Lobate

Form: Irregular, dry

Staining 

Staining is another method of classifying bacteria. Staining is done to facilitate the bacterial observation under microscope, as bacteria are generally minute and colourless when suspended in aqueous media. There are several staining method that can be done for bacteria classification: 

1. Simple staining - Simple staining using only one reagent, which produce distinctive contrast between the bacteria and its background. The common reagents used are methylene blue, crystal violet, and carbol fuchsin. 
2. Negative staining - Negative staining requires the use of acidic stain as the reagent. The acidic stain, with its negatively charged chromogen, will not penetrate into the bacteria because of the negative charge on the surface of bacteria. The common reagents used are India ink or nigrosin. 
3. Acid-fast staining - Acid fast staining is a staining method that is used to differentiate acid fast bacteria. Example of acid fast bacteria are the members of the genus mycobacterium, such as M. tuberculosis and M. leprae. 
4. Gram staining - Gram staining is a differential staining that requires the usage of four chemical reagents after heat fixation of bacteria. Gram staining divides the bacteria into two major groups: gram positive and gram negative. 

Gram staining procedure

1. Gently stain with crystal violet for one minute. Crystal violet acts as a primary stain that will stain all the present cells purple. Gently wash off the stain after a minute. 

2. Gently apply Gram's iodine for 1 minute. Gram's iodine acts as a mordant that increases the cells affinity for a stain. Gently wash off after a minute. 

3. Gently apply alcohol onto the slide until the slide decolourize. Alcohol acts as a decolourizing agent that also serves as a protein-dehydrating agent and a lipid solvent. Alcohol must be added until it runs almost clear with only a light tinge of blue. 

4. Counterstain with safranin for 45 seconds. Safranin is used to stain the cells that have been decolourized previously by the alcohol. Gently wash off after 45 seconds. 

Observation: 

Bacillus subtilis

Gram: Positive

Colour: Purple

E. coli

Gram: Negative

Colour: Pink

Pseudomonas

Gram: Negative

Colour: Pink

DISCUSSION

In gram staining, gram-positive bacteria will display purplish colouration, whereas gram-negative bacteria will display pinkish colouration when observed under light microscope. This is mainly affected by the peptidoglycan layer in cell wall of the bacteria. 

(Major Difference Between Gram-Positive and Gram-Negative Bacteria, n.d.)

Gram-positive bacteria have thicker layer of peptidoglycan in their cell wall, which retains the purple colour of the primary stain even after the application of alcohol wash. This causes the gram-positive bacteria to display purple colouration under light microscope.

On the other hand, the gram-negative bacteria have much thinner layer of peptidoglycan. This allows the primary stain to be removed when alcohol wash is applied. The cells instead retain pink colour of the counterstain (safranin). This causes the gram-negative bacteria to display pink colouration under light microscope.

REFERENCES

Cappuccino, J. G., & Welsh, C. T. (2017). Microbiology: a laboratory manual. Pearson Education.

Major Difference Between Gram-Positive and Gram-Negative Bacteria. (n.d.). Retrieved from https://byjus.com/biology/difference-between-gram-positive-and-gram-negative-bacteria/