Laboratory Techniques

Preliminary report / Presumptive ID (photo)

Typical Samples

• Pus swabs/fluid (Wounds)

• Other swabs (Nasal, throat)

• Sputum samples

• Aspirates (Joint, BAL)

• Urine samples

• Stool samples

• Medical devices (IUCD)

• Fluids (CAPD)

Aseptic Techniques (10)

• Keep caps and lids off the workbench - retain in hand

• Replace caps and lids as soon as possible

• Place agar media lids facing upwards

• After the plates are inoculated, the lids should be replaced

• Discard loops - open bench - dispose into sharps bin

• Keep samples away from the face when opening culture containers

• Wear appropriate PPE when handling cultures

• Open caps slowly in a microbiological safety cabinet - minimize aerosol production

• Autoclave and sterilize forceps or scissors before use

• Use disposable forceps or scissors - dispose into a sharps bin

Subculture from Solid to Liquid (4)

• Select a representative colony or colonies to sub-culture

• Use aseptic technique to transfer to an appropriate broth* with a sterile disposable loop

• Emulsify the organism using the inside surface of the container

• Gently agitate before incubation to distribute organisms throughout the broth

Inoculation Techniques

• Ensure that culture media are inoculated in a sequence that minimizes the risk of cross-contamination

• Liquid media should be inoculated after solid media when swabs and stools are examined

  • to avoid diluting the organisms present in the sample

• Liquid media should be inoculated first when processing fluid specimens

• Smears for staining are usually made after all culture media have been inoculated

• When the smear is required prior to culture, great care should be taken to avoid contamination

  • Do not place the loop back into the specimen after touching the slide

Inoculation Techniques Types (6)

• Streak Plate Method

  • Used to isolate a pure colony of a microorganism from a mixed culture

• Pour Plate Method

  • Used to estimate the number of viable microorganisms in a sample (TVC, TCC)

• Spread Plate Method

  • Used for enumeration of microbial populations, especially when counting colonies on the surface of the plate

• Stab Inoculation (Deep Inoculation)

  • Used to inoculate a solid medium to assess microbial growth under anaerobic or aerobic conditions - up&down

• Slant Inoculation

  • Used to grow a culture on a slanted agar surface for long-term storage of microbial cultures

    • + for growing microorganisms in a solid medium with a large surface area

  • solid media is in tube (glass container)

    • controlled environment + protected to not dry out

• Swab Inoculation

  • Used to obtain confluent growth on a surface medium

    • for susceptibility testing

    • for environmental / clinical sampling from surfaces

Streaking Agar Plates (3)

1. swap - create a pool

2. from pool outwards - pool up back down then up…

3. from those lines of streak outwards again and again

Uses:

• Growth on Culture Media

  • The streaking technique is used to grow and isolate microorganisms from a mixed culture on solid agar media, enabling colony isolation

• Automated Streaking

  • Use of automated equipment to perform streaking, which ensures consistent and reproducible isolation of colonies

  • improving laboratory efficiency and reducing human error

Streaking Agar Plates for urine sample steps

1. carry out count

2. check growth - if >100 (colony count) + <2 colonies → further testing

Optimal Atmospheric Conditions (photo)

• Strict Aerobes bacterial species

  • Require oxygen for growth - Aerobic

    • Haemophilus influenzae

• Strict Anaerobes

  • Cannot tolerate oxygen - anerobic

    • Bacteroides fragilis

• Facultative Anaerobes

  • Can grow in both aerobic and anaerobic environments - Aerobic & Anaerobic

    • Staphylococcus aureus

• Microaerophilic

  • Prefer low oxygen levels for optimal growth - Aerobic

    • Campylobacter jejuni

• Aerotolerant Anaerobes

  • Can survive in oxygen but do not use it for growth - Anaerobic

    • Clostridium perfringens

Inoculating from Broth Cultures

Transfer the broth culture to the solid medium

Swabbing Technique

Rotating of the technique results in confluent growth

Anaerobic Jar (photo!)

• sealed container

• Oxygen is eliminated through chemical reactions or gas-generation methods

• Palladium catalyst promotes the reaction of hydrogen with oxygen to form water, visible as droplets at the bottom of jar

• Indicators (chemical or biological) verify anaerobic conditions

  • Chemical indicator:

    • White: Reached anaerobic conditions

    • Blue: Anaerobic conditions not reached

  • Biological:

    • Strict Aerobe:

      • No growth

      • anaerobic conditions have been reached

    • Weak Aerobe

      • Growth

      • not reached

Anaerobic Incubator (4)

• Provides an oxygen-free environment for cultivating anaerobic bacteria

• Equipped with gas controls for nitrogen, hydrogen, and carbon dioxide levels

• Includes temperature regulation to optimize bacterial growth conditions

• Prevents oxygen exposure during incubation to maintain anaerobic conditions

CO₂ Generating Incubator (4)

• Provides a controlled environment with adjustable CO₂ levels to support the growth of microorganisms and cell cultures

• Maintains stable temperature, humidity, and CO₂ concentrations to mimic physiological conditions

• Used primarily for growing fastidious organisms requiring specific CO₂ levels

• Equipped with CO₂ sensors for precise monitoring and adjustments

Identification of Bacteria (9+6)

• Microscopy

  • Morphology - arrangement, cocci, bacilli

  • Staining reactions - positive/negative

• Cultural Characteristics

  • Colonial growth

    • Morphology

      • Size, shape of colony, edge, elevation

    • Changes brought to the culture medium

      • Haemolysis, pigmentation

    • Ability to grow on certain/specific media

      • Presence of bile, antibiotics

• Growth on special media

  • Selective (e.g., DCLS)

  • Differentiating (e.g., MSA)

• Biochemical characteristics

  • Carbohydrate fermentation

• Serological tests

• Analysis of metabolic end products

  • Identification of anaerobes

• Molecular biology techniques

• Growth characteristics*

Growth characteristics - Identification of Bacteria

• Rapidity (18 to 24 hours, 72 hours)

• Optimal atmospheric conditions

  • Anaerobes

    • Strict anaerobes (Bacteroides fragilis)

    • Aerotolerant (Clostridium perfringens)

  • Facultative anaerobes (Staphylococcus aureus)

  • Strict Aerobes (Haemophilus influenzae)

  • Microaerophilic (Campylobacter jejuni)

• Optimal temperature

  • Psychrophiles

  • Mesophiles

  • Thermophiles

Microscopy Morphology

Determines bacterial shape and arrangement

Microscopical Morphology (G+C) (photo!)

Description of Colonies (photo!)

Pleomorphism definition

Bacteria of the same species show different shapes and sizes

Bacterial Colony Characteristics (3)

• Umbonate

  • raised, convex center that looks like a dome

  • fx.: Staphylococcus aureus

• Filamentous

  • Thread-like, branching

  • fx.: Nocardia species*

• Draughtsman

  • Irregular, lobate edges resembling a draughtsman's pattern

  • Lobe-like morphology

  • fx.: Streptomyces species

Mucoid Colonies (3)

• Shiny, sticky, due to polysaccharide capsule

• Helps in virulence and immune evasion

• fx.: Klebsiella species

Swarming Colonies (4)

• Concentric rings formed by flagellar motility

• Enhanced black edges for contrast

• Associated with rapid movement across agar

• fx.: Proteus species

Haemolysis definition

The breakdown of red blood cells, which can be observed when bacteria produce enzymes that lyse red blood cells in agar media

• Alpha-haemolysis (α-haemolysis)

  • Partial breakdown of red blood cells, creating a green or brown discoloration around the colony

  • Fx.: Streptococcus pneumoniae

• Beta-haemolysis (β-haemolysis)

  • Complete breakdown of red blood cells, leading to a clear zone around the colony due to the full lysis of the cells

  • Fx.: Streptococcus pyogenes

green - partial distraction
Beta - complete distraction