Microbiology Lab Exam 1

• LAB EXAM 1

Microscopy

• Objective lenses: magnify the specimen.
• Condenser: Focuses light.
• Light intensity: adjusts current to lamp.

Aseptic Technique

• Purpose: prevent/reduce transmission.
• Sterilize loops until red, cool, and allow to grow.
• Open tube.
• Mix with loop and collect sample.
• Flame tube mouth and cap.
• Transfer sample.
• Flame loop til red hot.

Ubiquity of Organisms

• Types of media: broth culture, plates, agar slants and nutrient broths.
• Defined media has an exact chemical composition that is known; it differentiates batch to batch.
• Turbid: cloudy or hazy.
• Pellicle: thin film at the top.
• Flocculent: clumps/flakes throughout.
• Sediment: bacterial growth that collects at the bottom.

Simple Stain

• What is simple stain? The use of a positively charged chromophore that is absorbed by the negatively charged cell when they come into contact.
• Requires heat fixation.
• Aseptically transfer loopfull of specimen onto slide.
• Sterilize loop.
• If specimen is concentrated, use the specimen and mix with inoculating neural water.
• Air dry.
• Pass slide through flame 2-3x to heat fix it.
• Cover smear with stain, let sit for 1 minute.
• Pat dry using biblious paper.
• Observe up to oil.
• Bacteria will appear color of dye.
• Basic dyes have positive charge which are attracted and absorbed by the bacteria.
• Examples of basic dyes are: Methylene blue, crystal violet, safranin.

Negative Stain

• What is negative stain? The use of a negatively charged stain that is repelled by negatively charged bacteria when they come into contact. Cell remains clear and background is stained.
• Doesn't require heat fixation.
• Add drop of dye to slide.
• Add/mix specimen on top of drop of dye.
• Spread mixed drop across slide using another clean slide, starting in the middle and dragging it to the drop of dye and then back to the opposite end of the slide.
• Air dry for 5-10 mins.
• Observe.
• Background will be dyed and bacteria appear clear.
• Acidic dyes have a negative charge chromophore that is repelled by negatively charged bacteria.
• Examples of reagents: Nigrosine, Congo Red.

Staining

• Differential stain: using more than 1 stain to differentiate organisms. NO HEAT FIX.

Gram Staining

• Gram (+) stains purple.
  • Thick peptidoglycan.
  • Teichoic acids.
• Gram (-) stains pink.
  • Thin peptidoglycan.
  • Outer membrane.
  • LPS.
  • Periplasmic space.
  • Porins.
• Reagents: crystal violet, iodine (mordant), decolorizer and safranin.
• Protocol:
  • Crystal violet 1 minute then water rinse.
  • Iodine (mordant) 1 minute then water rinse.
  • Decolorizer 10 seconds then water rinse.
  • Safranin 45 seconds.
• Clinical application: can help narrow down antibiotic choice for certain infections.

Structural Stains

• Endospore Staining: Used with heat.
• Flagella Stain: Need to add more into the cell so we make them thicker and then stain it using carbolfuchsin.
• Capsule Stain: Capsules remain unstained.
  • Background stains with nigrosine (negative stain).
  • Differential and simple.
  • Polysaccharide layer.

Acid Fast Bacteria

• How to tell acid-fast bacteria from non-AF bacteria: Have Mycolic acid (waxy lipid) in walls that resist decolorization, leaving the walls stained.
• Stain with carbolfuchsin, decolorize, and counterstain with methylene blue.
• Mycobacterium Leprae causes leprosy and tuberculosis causes TB.
• Used to Identify Oocysts (thick outer wall that serves as protection) found in cocci parasites.
• Acid Fast is a differential stain, that's used to ID mycobacteria.
• Reagents: carboulfuchsin (high phenol will be used), heat (sometimes), alcohol for decolorizer, methylene blue for counterstain.

Ziehl-Neelsen vs Kinyoun

• Ziehl-Neelsen (Z.N) uses heat as mordant and Kinyoun (K.M) uses highly concentrated carbolfuchsin and higher phenol as mordant. No Heat.
• Used to ID pathogens in Mycobacterium genus.

Streak Plate

• Quadrant-streak plate: 4 sections streak while sterilizing.
• T-streak plate: 3 sections streak while sterilizing between each sections.
• Aseptically collect sample.
• Sterilize loop.
• Repeat step and gently drag loop into the previous streak.
• Continue until last streak is reached.
• Diluted sample is needed.
• Properly spread the sample on the media to get isolated colonies.

Isolation Technique

• Spread plate, pour plate
• Spread Plate: Requires diluted sample to begin.
• Pour Plate: Useful for counting colonies; a diluted culture is added to molten agar and sterile plate.

  • 25 TFTC

  • 30-300 valid colony count.

Special Media

• Selective media: includes CNA, MSA, MAC 100. Selective agent can select for Gram + bacteria/alat.
• Differential media: is used to distinguish one species of bacteria from another.
• EMB selects for Gram - bacteria and distinguishes based on lactose fermentation.

Anaerobic Growth

Incubate plate, place petri dish on plate spinner.

• Using new pipette, transfer sample from serial dilution to petridish.
• Use sterile spreader to spread sample on dish while spinning plate.

Reducing Media

• Reducing agents (fluid thioglycolate) are added to the media to combine with leftover O2 and convert it.
• Oxygen indicators like resazurin are added to indicate the presence of O2.
• Pink = O2.
• Mix of special gasses that allows anaerobic growth.
• Catalyst removes leftover O2 and convert it.

Oxygen Requirements

• Obligate Aerobes: Need O2 to grow.
• Obligate Anaerobes: Need O2 free.
• Facultative Anaerobes: Grow with or without O2.
• Microaerophiles: Need a limited concentration of O2 to grow.

Catalase

• Catalase is an enzyme that breaks down hydrogen peroxide (H2O2) into H2O and O2
• Positive reaction (catalase): bubbles (O2) form after adding hydrogen peroxide.
• Negative reaction (catalase): no bubbles produced after adding hydrogen peroxide.

Difference between spread pour and streak

• Spread plate is poured on top of surface and colonies grow on the surface of the media.