Bacteriology Review: Gram Positive and Negative Microorganisms

Bacteriology: Review Notes

Characteristics of Bacteria

• Bacteria are single-cell prokaryotic microorganisms that divide by binary fission.
• They are 0.5-1 micron in size, comprising mostly water (70%), carbohydrates, proteins, lipids, and enzymes.

Gram Staining

• Gram staining is a method employed in the identification of bacteria in the laboratory.
• The method includes crystal violet, Gram's iodine, and safranin.
• Teichoic acid is present in Gram-positive bacteria, while outer membrane with lipopolysaccharides (LPS) and porins is present in Gram-negative bacteria.

Classification of Bacteria

• Prokaryotes can be classified into three groups: Eubacteria (true bacteria), Cyanobacteria (blue-green algae), and Archaebacteria (survive in extreme environments).
• Gram-positive bacteria include Bacillus, Corynebacterium, Erysipelothrix, Lactobacillus, Listeria, Mycobacterium, and Nocardia.
• Gram-negative bacteria include Acinetobacter, Aeromonas, Alcaligines, Bordetella, Brucella, Enterobacteriaacea, Francisella, Legionella, Pasteurella, Pseudomonas, and Vibrio.

Bacterial Cell Structure

• The cell wall is responsible for the shape of the bacteria and contains peptidoglycan (also known as murein layer).
• The cytoplasmic membrane is the site of energy production among prokaryotes and is selectively permeable.
• Mesosomes are points of attachment of chromosomes, while inclusions include Much's granules (lipids), Babe-Ernst granules (polyphosphates), and ribosomes.

Acid-Fast Staining

• Acid-fast staining is used to identify Mycobacterium tuberculosis.
• The Ziehl-Neelsen stain uses carbol fuchsin, acid alcohol, and methylene blue.
• Modifications of the Ziehl-Neelsen stain include the Pappenheim stain and the Baumgarten stain.

Motility

• Motility can be observed through hanging drop preparation (HDP) or wet mount.
• Methods to determine motility include HDP, SIM (Sulfide Indole Motility), staining the flagella, serological tests, swarming tests, and fluorescence tests.

Virulence Factors

• Virulence factors include adherence factors (pili or fimbriae), antiphagocytic factors (capsule), enzymes (hyaluronidase), and toxins (endotoxin or exotoxin).

Microscopy Types

• Types of microscopy include bright-field microscopy, dark-field microscopy, fluorescent microscopy, and electron microscopy.

Bacterial Growth Factors

• Bacterial growth factors include nutrients (carbon source, nitrogen source, minerals, and salts), oxygen and carbon dioxide availability, and temperature.
• Obligate aerobes require ambient air, while obligate anaerobes grow without oxygen. Facultative anaerobes can survive without oxygen, and microaerophilic bacteria require reduced oxygen.### Specimen Collection and Transport
• Specimens for bacteriology should be collected properly to prevent contamination with normal skin flora.
• Use of swabs: Cotton or Dacron/Ca alginate swabs are used for collection.
• Transport media:
  • Stuart's medium: for gonococci, contains anaerobic salt solution.
  • Cary and Blair medium: for V. cholerae, S. typhi, and Yersinia pestis.
  • Amies medium: for Vibrio sp., contains charcoal to adsorb bactericidal/static substances.
  • Glycerol saline medium: for enteric fever bacilli (Salmonella).

Culture Media

• Classification of culture media:
  • According to consistency: liquid, semi-solid, and solid.
  • According to composition: synthetic/defined, non-synthetic, and tissue culture media.
• Types of culture media:
  • Enriched media: support the growth of fastidious organisms.
  • Selective media: promote growth of desirable organisms and inhibit others.
  • Differential media: distinguish between different organisms based on their characteristics.
  • Special or specific media: support the growth of specific organisms.

Bacteriological Examination

• Interpretation of culture results:
  • CFU/ml: number of colonies per milliliter of specimen.
  • Significant bacteriuria: ≥105 CFU/ml.
  • Doubtful bacteriuria: 103-105 CFU/ml.
  • Possible contaminant: <103 CFU/ml.
• Types of colonies based on serologic characteristics:
  • Mucoid: glistening and water-like in appearance.
  • Smooth: homogenous and uniform without appearing as liquid or mucoid.
  • Rough: granulated and rough.

Identification of Microorganisms

• Selective/Differential media for gram-negative enteric bacilli:
  • Eosin-Methylene Blue (EMB) agar: lactose fermenters produce pink-purple colonies.
  • MacConkey agar: lactose fermenters produce pink colonies.
  • Hektoen Enteric Agar: lactose fermenters produce yellow colonies with black centers.
• Selective/Differential media for other microorganisms:
  • Salmonella-Shigella Agar: for Salmonella and Shigella.
  • Mannitol Salt Agar: for Staphylococcus sp.
  • Thiosulfate Citrate Bile Salt Sucrose (TCBS) agar: for Vibrio sp.

Antimicrobial Susceptibility Testing (AST)

• Steps in performing AST:
  1. Inoculate Mueller Hinton agar/broth with test organism.
  2. Apply antibiotic discs.
  3. Invert and incubate at 37°C for 16-18 hours.
  4. Measure the zone of inhibition.
  5. Interpret susceptibility from the standard chart.
• Possible sources of errors in AST:
  • Mixed culture
  • Inoculum too light or heavy
  • Improper storage of discs
  • Deterioration of turbidity standard or control strains
  • Reading and clerical errors

Biological Activities of Microorganisms

• Enzyme production: coagulase, catalase, etc.
• Production of hemolysin:
  • α (alpha) hemolysin: incomplete or partial hemolysis.
  • β (beta) hemolysin: complete hemolysis.
  • γ (gamma) hemolysin: no hemolysis.
• Production of pigment in bacteria:
  • Serratia marcescens: red (prodigiosin).### Bacteriology Review Notes

Pigment Production

• Chromobacterium violaceum: produces violet/purple pigment
• Pseudomonas aeruginosa: produces blue pigment (pyocyanin) and green pigment (pyoverdin)
• Pseudomonas fluorescens: produces yellow-green pigment (pyoverdin)
• Staphylococcus aureus: produces yellow-golden pigment
• Staphylococcus epidermidis: produces white pigment
• Actinomyces: produces silver pigment
• Micrococcus roseus: produces pink pigment
• Prevotella melaninogenica: produces black pigment
• Sarcina aurentiaca: produces orange pigment

Biochemical Reactions

• Catalase test: differentiates gram-positive and gram-negative organisms
  • Principle: catalase breaks down hydrogen peroxide (H2O2) to oxygen (O2) and water (H2)
  • Media: hydrogen peroxide (H2O2)
  • Result: (+) bubble formation, (-) no bubble formation
• Coagulase test: differentiates Staphylococcus aureus from other Staphylococcus species
  • Principle: coagulase breaks down fibrinogen to form a clot
  • Media: plasma
  • Result: (+) clot formation, (-) no clot formation

Streptococci

• Bacitracin disk test: differentiates group A beta-hemolytic Streptococci (Streptococcus pyogenes) from other beta-hemolytic Streptococci
  • Principle: Streptococcus pyogenes is susceptible to low doses of bacitracin
  • Media: Taxo A-Bacitracin disk
  • Result: (+) zone of inhibition, (-) no zone of inhibition
• Optochin disk test: differentiates alpha-hemolytic Pneumococci (Streptococcus pneumoniae) from other alpha-hemolytic Streptococci
  • Principle: Streptococcus pneumoniae is sensitive to optochin
  • Media: Taxo P-Optochin disk
  • Result: (+) zone of inhibition, (-) no zone of inhibition

Test for Gram-Negative Organisms

• Oxidase test: differentiates Neisseria and Pseudomonas from other gram-negative bacteria
  • Principle: oxidase breaks down tetramethyl-p-phenylenediamine to form a colored complex
  • Media: Kovac's reagent
  • Result: (+) dark purple color, (-) no color
• Indole test: differentiates gram-negative bacteria based on their ability to split indole from tryptophan
  • Principle: kovac's reagent detects the presence of indole
  • Media: tryptophan broth
  • Result: (+) red color, (-) no color

Carbohydrate Fermentation

• CHO (carbohydrate) fermentation test: differentiates bacteria based on their ability to ferment carbohydrates
  • Principle: fermentation of simple sugars serves as the main source of energy for microorganisms
  • Media: various carbohydrate media (e.g., glucose, lactose, etc.)
  • Result: (+) acid production, (-) no acid production

Nitrogen Metabolism

• Methyl red test: differentiates bacteria based on their ability to produce acidic or alkaline metabolites
  • Principle: methyl red indicator detects the presence of acidic metabolites
  • Media: Clark and Lubbs Dextrose Broth
  • Result: (+) red color, (-) yellow color
• Voges-Proskauer test: differentiates bacteria based on their ability to produce acetoin or acetylmethylcarbinol
  • Principle: Voges-Proskauer reagent detects the presence of acetoin or acetylmethylcarbinol
  • Media: Clark and Lubbs Dextrose Broth
  • Result: (+) red color, (-) yellow color

Decarboxylase/Dihydrolase Test

• Decarboxylase test: differentiates bacteria based on their ability to decarboxylate or hydrolyze amino acids
  • Principle: decarboxylase breaks down amino acids to form amines
  • Media: LOA (Lysine Ornithine Arginine) medium
  • Result: (+) red or purple color, (-) yellow color

Molecular Diagnosis

• Polymerase Chain Reaction (PCR): amplifies specific DNA targets
  • Principle: PCR uses primers to amplify specific DNA sequences
  • Media: PCR reagents and template DNA
  • Result: (+) amplification of target DNA sequence, (-) no amplification
• Loop-Mediated Isothermal Amplification (LAMP): amplifies specific DNA targets
  • Principle: LAMP uses a single tube technique for amplification
  • Media: LAMP reagents and template DNA
  • Result: (+) amplification of target DNA sequence, (-) no amplification
• MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization-Time of Flight) mass spectrometry: identifies microorganisms based on their molecular mass
  • Principle: MALDI-TOF uses a laser to ionize molecules, which are then separated based on their mass-to-charge ratio
  • Media: MALDI-TOF matrix and sample
  • Result: (+) identification of microorganism, (-) no identification