PRELIMS

MLS 411-LAB Bacteriology Comprehensive Reviewer

PART 1: GRAM STAIN

Overview

• Developed in 1884 by Hans Christian Gram

• Classifies bacteria into Gram-positive and Gram-negative

Reagents (VIAS)

Steps

1. Apply crystal violet → heat-fixed smear turns purple

2. Apply iodine → both G+ and G- appear dark violet

3. Apply alcohol/acetone → removes purple from some bacteria

4. Apply safranin → counterstains decolorized cells pink/red

Results

• Gram-positive → retains dark violet/purple

• Gram-negative → loses purple, takes up safranin (pink/red)

Microscopic Examination

Gram Stain Quantities

Errors in Gram Staining

Smear Prep

• Non-sterile: Sputum, urine, oral swab, wound swab → smear thinly (monolayer)

• Presumed sterile: CSF, body fluids → use cytocentrifuge/cytospin

Bacterial Morphologies (Key Examples)

• Gram-positive cocci in clusters

• Gram-positive cocci in chains or pairs

• Gram-negative diplococci (intracellularly — urethral smear)

• Gram-positive rod with endospores

• Gram-negative rod (thin, thick, fusiform)

• Gram-positive rod diphtheroid / boxcar / filamentous

PART 2: ACID FAST STAIN (AFS)

Principle

• Primary stain binds to mycolic acid in cell walls of acid-fast bacteria

• Retained after decolorization with acid-alcohol

• Carbolic acid strengthens the bond between mycolic acid and cell wall

• Heat acts as a physical mordant — opens up the thick cell wall

• Commonly used when TB (tuberculosis) is suspected

Results

Reagents

1. Carbolfuchsin — primary stain

2. Heat — mordant (Ziehl-Neelsen) / Tergitol (Kinyoun)

3. Acid-alcohol (3% HCl + 95% ethanol) — decolorizer

4. Methylene blue or Malachite green — counterstain

Lab Steps (Ziehl-Neelsen)

1. Apply carbolfuchsin; heat until steam → leave 10 minutes

2. Cool and wash with water

3. Decolorize with acid-alcohol → 25–30 seconds

4. Apply methylene blue → 10 seconds

5. Examine under 1000x OIO — AFB appear red/pink on blue background

Lab Manual Steps (Simplified)

1. Apply carbolfuchsin to saturated paper; heat 5 min in exhaust hood

2. Cool; rinse with water 30 sec

3. Decolorize with acid-alcohol until pink (10–30 sec)

4. Rinse with water 5 sec

5. Counterstain with methylene blue 20–30 sec

6. Rinse 30 sec → blot dry with bibulous paper

Acid Fast Staining Methods

National Standard Reporting Scale

Causes of False Readings in AFS Smears

PART 3: BACTERIAL CULTURE MEDIA

Cultivation

Process of growing microorganisms by collecting bacteria from an infection site and growing them in laboratory media containing nutrients (proteins, carbs, metals, minerals, buffers).

• Fastidious — require complex nutrients (growth factors, vitamins)

• Non-fastidious — basic nutritional needs

Media Classifications

Media with Dual Functions

• MacConkey Agar → Differential AND Selective

• Sheep Blood Agar → Supportive AND Differential (most used supportive medium in diagnostic bacteriology)

Preparation of Culture Media (Key Steps)

1. Read label instructions carefully

2. Measure agar + distilled water into a flask

3. Flame-sterilize glass rod

4. Heat with constant stirring; simmer 1 minute — DO NOT BOIL

5. Plug with cotton + foil; label (section, group, media, date)

6. Submit for autoclaving

7. After autoclaving, let stand briefly before dispensing

8. Pour 1/8" to 1/4" deep into sterile petri dishes

9. Cool at room temperature until set

Storage

• Store agar plates upside down in the refrigerator

• Do NOT freeze

• Upside down = prevents condensation from dripping onto agar surface (prevents contamination/swarming)

PART 4: BACTERIAL CULTIVATION TECHNIQUES

Key Definitions

• Bacteria — microscopic organisms; visible macroscopically as colonies when mass multiplication occurs

• Cultivation — planting/seeding of a sample for bacterial growth

• Subculturing — transferring microorganisms from one medium to another

Why Aseptic Technique?

• Microorganisms are found everywhere (ground, air)

• Prevents contamination of subcultures by unwanted microbes

Aseptic Transfer Steps

1. Heat wire loop until red hot

2. Cool in air briefly

3. Uncap culture tube

4. Heat the mouth of the tube

5. Gather the inoculum with the loop

6. Reheat the mouth of the tube

7. Recap the tube

Inoculation Methods

Streak Plate Method (Tube to Plating)

1. Label plate (name, date, "BC-SP")

2. Aseptically obtain a loopful; streak into Area 1

3. Sterilize loop; cool on agar edge

4. Cross previous streak once → streak into Area 2

5. Repeat for Area 3 and 4

   • 4th streak must NOT touch earlier streaks

6. Invert plates; incubate 24–48 hours at appropriate temperature

7. After incubation → refrigerate in inverted position

After Incubation — Colony Observation

Describe colonies by:

• Size (measure in mm)

• Color

• Characteristics (shape, surface, opacity, elevation, margin)

Random Surface Samples

• Label plate "RS-SP"

• Swipe a sterile swab on surfaces exposed to dirt

• Streak onto NA plate

• Incubate 24–48 hours, then refrigerate inverted

• Examine for well-isolated colonies

QUICK COMPARISON: GRAM STAIN vs. ACID FAST STAIN