Bacteriology Review: Gram Positive and Negative Microorganisms

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What are the characteristics of Mucoid colonies?

Glistening and water-like in appearance

Describe the appearance of Smooth colonies.

Homogenous and uniform without appearing as liquid or mucoid

What is the characteristic of Rough colonies?

Appear granulated and rough

What are the types of colonies based on serologic characteristics?

All of the above

Solid culture media can be categorized into plated and tubed types.

True

What process do bacteria use to divide?

binary fission

All cocci are gram positive except Neisseria, Branhamella, and Veilonella.

True

Which special stain is used to demonstrate the capsule of bacteria?

Hiss stain

The _ divides bacteria by defining their shape and is mainly composed of peptidoglycan.

cell wall

Match the following bacterial structures with their functions:

Teichoic acid (Mg++) = Gram positive bacteria Outer membrane (LPS) = Gram negative bacteria Capsule = Prevents phagocytosis Flagella = For locomotion

What process is used to stain acid-fast organisms?

acid fast staining

What type of movement do bacteria exhibit in liquid medium?

Brownian movement/False movement/Molecular movement/On-the-spot vibratory movement

Which factors are considered virulence factors in bacteria? (Select all that apply)

Enzymes

Exotoxins are released by __________ cells, while endotoxins are released upon lysis of cells.

living

Endotoxins are mostly released by Gram-negative bacteria.

True

Match the following microscopy types with their descriptions:

Bright-field microscopy = Standard microscopy with a white background Dark field microscopy = Microscopy where specimens appear bright against a dark background Fluorescent microscopy = Microscopy using fluorescent dyes for visualization Electron microscopy (TEM/SEM) = High-resolution microscopy using electrons for imaging

What are the possible sources of errors in susceptibility testing?

  1. Use of mixed culture
  2. Inoculum too light
  3. Inoculum too heavy
  4. Too much moisture on agar
  5. Very dry agar surface
  6. Improper storage of disc

Increased cations like Mg++ and Ca++ can cause increased resistance of P.aeruginosa to aminoglycosides.

True

A positive catalase test results in the formation of _ bubbles.

oxygen

Which of the following colors is associated with Staphylococcus aureus?

Yellow-golden

Match the following hemolysis types with their descriptions:

α (alpha) = Incomplete or partial hemolysis β (beta) = Complete hemolysis/clear zone γ (gamma) = No hemolysis

What is the principle behind the Decarboxylase/Dihydrolase test?

Some organisms decarboxylate or hydrolyze an amino acid to form an amine, resulting in an alkaline pH range.

Which color indicates a positive result in the Citrate test?

Prussian Blue

Lysine decarboxylase test is used to detect the ability of organisms to convert lysine into cadaverine.

True

ONPG test is based on the utilization of _______.

lactose

Match the molecular diagnosis technique with its description:

Polymerase Chain Reaction (PCR) = unknown sample -> extraction of DNA/RNA -> amplicons (PCR product) -> Identification LAMP (Loop mediated isothermal amplification) = single tube technique for the amplification of DNA MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization-Time of Flight) = mass spectrometry ionization technique using laser energy absorbing matrix

Study Notes

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

Review notes on bacteriology, covering gram negative and positive cocci and bacilli, including aerobic and anaerobic microorganisms.

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