Ways to Identify an Unknown Microbe Staph & Strep

Identifying Microbes

• Signs and symptoms can sometimes be used to diagnose a microbe or infection.
  • Signs are objective and measurable, like fever or rash.
  • Symptoms are subjective and describe a person's feelings or sensations, like pain or fatigue.

Patient History and Public Health

• Patient information, such as age, job, medical history, vaccination status, and current outbreaks, can help identify a microbe or infection.

Culture Media

• Provides nutrients and the right environment for microbes to grow.
• Can be solid, liquid, or semi-solid.
• Solid media contains agar for solidification, allowing for colonies to grow on the surface or be stabbed into the agar.
• Liquid media shows growth as turbidity, with no visible colonies.

General-Purpose Media

• Contains a mixture of nutrients to grow a wide range of microbes.
• Examples include nutrient broth/agar and trypticase soy broth/agar.

Enriched Media

• Contains additional complex organic substances like blood, serum, or growth factors.
• Used to grow fastidious microbes.
• Examples include blood agar and chocolate agar.

Selective Media

• Contains substances that inhibit the growth of certain microbes, selecting for the growth of specific ones.
• Helps isolate certain microbes from a mixed culture.
• Examples include mStaph Broth (high salt concentration selects for halotolerant microbes) and Sabouraud's Agar (low pH inhibits bacterial growth, selecting for fungus).

Differential Media

• Allows several types of microbes to grow but distinguishes between them through visible differences.
• Helps identify microbes by differentiating them based on their growth characteristics.
• Examples include urea broth (pH indicator differentiates microbes based on urease enzyme presence), and Chromagar (different dyes turn bacterial colonies specific colors to identify urinary pathogens).

Blood Agar

• Enriched media containing blood to support the growth of fastidious microbes.
• Differential media as it shows hemolysis patterns (how microbes affect blood).
  • Alpha hemolysis: incomplete lysis, brownish-green halos around growth.
  • Beta hemolysis: complete lysis, clear halos around growth.
  • Gamma hemolysis: no lysis, growth doesn't affect the blood.

Mannitol Salt Agar (MSA)

• Both selective and differential media.
• High salt concentration (selective) for halotolerant microbes.
• Mannitol and pH indicator (differential), where mannitol fermenters turn media yellow and non-fermenters leave it pink.

Differential Staining

• Uses two dyes to differentiate between cell types or parts.
• Steps:
  • Make a heat-fixed smear.
  • Primary stain.
  • Mordant.
  • Decolorizer.
  • Secondary or counterstain.

Bacterial Cell Shapes

• Coccus (cocci): spherical.
• Bacillus (bacilli): rod-shaped.
• Vibrio: comma-shaped.
• Spirillum: spiral-shaped.
• Spirochete: long, helical, flexible.

Bacterial Arrangements

• Single: individual cells.
• Diplo: pairs.
• Strepto: chains.
• Tetrad: groups of four.
• Sarcina: cube-like groups of eight.
• Staphylo: clusters.

Gram Stain (Differential Stain)

• Uses four steps to differentiate bacteria based on cell wall structure.

• Starts with a heat-fixed smear.

• Steps:

  • Primary stain: Crystal Violet.
  • Mordant: Iodine.
  • Decolorizer: Ethanol.
  • Counterstain: Safranin.

• Interpretation:

  • Purple: Gram-positive bacteria (thick peptidoglycan layer retains the crystal violet stain).
  • Pink: Gram-negative bacteria (thin peptidoglycan layer allows the stain to be washed away during decolorization, so the counterstain safranin is visible).

Ways to Identify a Microbe

• Signs are objective, measurable, and observable characteristics, such as fever or rash.
• Symptoms are subjective, feelings or sensations a person has, such as pain or fatigue.
• Epidemiology involves factors like patient's age, job, medical history, vaccination status, current outbreaks in the area, and risk factors specific to the location and/or patient's background.

Culture Media

• Purpose: Provide the nutrients and environment needed for microbial growth.
• Solid Media: Contains agar as a solidifying ingredient, allowing colonies to grow on the surface and/or be stabbed down into the agar.
• Liquid Media: Identified by turbidity (cloudiness), no colonies are present, and bacteria grow in the liquid.

Types of Culture Media

• General-Purpose Media: Contains a mix of nutrients designed to grow a broad range of microbes. Examples: Nutrient Broth/Agar, Trypticase Soy Broth/Agar.
• Enriched Media: Contains additional complex organic substances like blood, serum, and growth factors, required for growing fastidious microbes. Examples: Blood Agar, Chocolate Agar.
• Selective Media: Contains something to inhibit the growth of some microbes, selecting for the growth of specific ones. Helps to isolate a microbe from a mixed culture. Examples: mStaph Broth (high salt concentration for halotolerant microbes), Sabouraud’s Agar (low pH for fungus growth).
• Differential Media: Allows several types of microbes to grow but shows a visible difference between them, differentiating between growing microbes and aiding in identification. Examples: Urea broth (pH indicator for urease-producing microbes), Chromagar (dyes that turn colonies distinct colors for urinary pathogen identification).

Blood Agar: Enriched and Differential

• Enriched with blood, allowing fastidious microbes to grow.
• Differential due to hemolysis:
  • Alpha hemolysis: incomplete lysis (brownish green halos around growth).
  • Beta hemolysis: complete lysis (clear halos around growth).
  • Gamma hemolysis: no lysis (growth doesn’t affect the blood).

Mannitol Salt Agar: Selective and Differential

• Selective: High salt concentration allows only halotolerant microbes to grow.
• Differential: Mannitol fermenters turn the media yellow, while non-fermenters keep the media pink.

Differential Staining: Gram Stain

• Purpose: Uses two dyes to differentiate between cell types or parts.
• Steps:
  • Heat-fixed smear.
  • Primary stain: Crystal Violet.
  • Mordant: Iodine.
  • Decolorizer: Ethanol.
  • Counterstain: Safranin.
• Interpretation:
  • Purple: Gram Positive.
  • Pink: Gram Negative.

Bacterial Cell Shapes and Arrangements

• Shapes: Coccus (cocci), Bacillus (bacilli), Vibrio, Spirillum, Spirochete.
• Arrangements: Single, Diplo, Strepto, Tetrad, Sarcina, Staphylo.