AM - Practical Images

The procedure for preparing a smear from solid agar involves several precise steps to ensure clear microscopic observation.
Preparation Steps:
- Using a cooled sterile loop, place one small loopful of sterile water inside a previously prepared wax circle on a slide.
- Sterilize the loop again and, once cool, touch a SMALL amount of a single isolated colony.
- Emulsify the colony into the drop of water on the slide.
- Mix gently to create a smooth, thin suspension.
- Spread the suspension evenly within the wax circle.
- Allow the smear to air dry completely.
- Critical Requirement: The smear must be thin and consist of a single layer of cells. Thick smears are known to stain unevenly and are difficult to interpret.
Heat Fixing:
- Once the smear is dry, pass the slide (with the smear side up) quickly through a Bunsen flame $3$ times.
- Caution: Do not overheat the slide, as excessive heating damages cell structure.
Safety Note: Fixed smears may not be sterile and should be treated as contaminated.

Perform Gram stains on three experimental smears and one Gram control smear, following timing instructions carefully.
Step-by-Step Methodology:
- Flood the smear with crystal violet for $30\,s$ .
- Gently rinse the slide with water.
- Flood with Lugol’s iodine for $30\,s$ .
- Rinse again with water.
- Decolourize with alcohol-acetone for $2-5\,s$ . Take care not to over-decolourise; once the slide runs clear, rinse with water immediately.
- Flood with safranin for $30\,s$ .
- Rinse with water and blot dry carefully.
Observing and Recording Results:
- Individual Bacterial Cell Description:
  - Gram Reaction: Gram-positive bacteria appear purple, while Gram-negative bacteria appear pink/red.
  - Shape: Identify if the cells are cocci or bacilli.
  - Arrangement: Observe if the cells are in chains, clusters, pairs, or single.
  - Approximate Size: Record the estimated size of the cells.
  - Quality: Note the quality of the staining.
- Final Result Report: Formulate as a two-part statement (e.g., Gram-positive cocci or Gram-negative bacilli).

This procedure requires specific safety protocols due to toxic vapors.
Safety Note: Heating carbol fuchsin releases phenol vapour, which is toxic if inhaled. This procedure must be performed in the chemical fume hood with the sash lowered.
Methodology:
- Transfer slides to the chemical fume hood and place them on the heating block.
- Flood the smear with carbol fuchsin and gently heat for $5\,min$ .
- Ensure the smear does not dry out; add more stain if necessary.
- Rinse gently with tap water while still in the chemical fume hood.
- Move slides to the laboratory bench sinks.
- Decolourize with $3\%\,\text{acid alcohol}$ for $2-3\,min$ , or until washings run clear.
- Rinse thoroughly with water.
- Counterstain with $0.3\%\,\text{methylene blue}$ for $2\,min$ .
- Rinse with water and blot dry.

Blood agar is used as an enriched and differential medium. It supports a wide range of organisms and differentiates them based on haemolytic activity (the breakdown of red blood cells by bacterial enzymes).
Types of Haemolysis:
- Beta: Complete clearing around colonies due to complete lysis of red blood cells.
- Alpha: Partial haemolysis producing a green or brown discolouration.
- Gamma: No haemolysis; there is no visible change in the agar.
Atmospheric Conditions:
- Bacterial growth is influenced by oxygen availability. Plates are incubated under Aerobic, Anaerobic, and Carbon Dioxide ( $CO_2$ ) enriched conditions.
- A throat swab normally contains a mixed microbial population.
Activity Goals:
- Compare bacterial growth across different atmospheric conditions.
- Identify and describe haemolysis on blood agar.
- Recognise mixed cultures.
- Link colony morphology and growth conditions to potential bacterial identities.

Oxidase Test:
- Place filter paper in a Petri dish and add $1\,\text{drop}$ of oxidase reagent.
- Use a toothpick to smear a small amount of colony onto the reagent.
- Interpretation: Positive result is a dark purple/blue colour within $10-30\,s$ . Negative result is no colour change.
API Strip Interpretation:
- CIT (Citrate): Positive is distinctly green/blue.
- GEL (Gelatinase): Positive involves the actual breakdown of gelatin.
- Carbohydrate Tests (GLU onwards): Positive is yellow (acid production); Negative is green/blue.
Catalase Test:
- Place two drops of $H_2O_2$ on a slide.
- Mix colonies into individual drops.
- Interpretation: Positive result is immediate bubbling ( $O_2\,\text{gas}$ ); negative is no bubbles.
- Example: Staphylococcus epidermidis is catalase-positive; Streptococcus agalactiae is catalase-negative.
Coagulase Test:
- Place two drops of rabbit plasma on a slide.
- Emulsify colonies using sterile toothpicks.
- Interpretation: Positive result is visible clumping (coagulation); negative is a smooth suspension.
- Example: Staphylococcus aureus is coagulase-positive; Staphylococcus epidermidis is coagulase-negative.

Gram-Positive Cocci Identification Path:
- Catalase Positive ( $+\text{bubbles}$ ) = Staphylococcus genus —> Coagulase Test.
  - Coagulase Positive ( $+\text{clumps}$ ) = S. aureus.
  - Coagulase Negative ( $-\text{no clumps}$ ) = S. epidermidis.
- Catalase Negative ( $-\text{no bubbles}$ ) = Streptococcus genus.
Gram-Negative Bacilli Identification Path:
- Oxidase Positive ( $+\text{colour change}$ ) = non-Enterobacteriaceae (use API20NE strip).
- Oxidase Negative ( $-\text{colour change}$ ) = Enterobacteriaceae (use API20E strip).