Lab Exam 1 Review - Microbiology

• Biosafety level (BSL)


  Goes from 1 to 4 based on the likelihood for a microbe to cause diseases and how virulent (harmful) the microbe can be
• Our lab is treated as a BSL-2 lab
• Proper attire in the lab

  • Shoes should not expose any skin on the foot and be closed-toe and closed-heel
  • Shoulders, chest, and abdomen must be covered
  • Wear a lab coat when indicated by the instructor
  • Tie long hair back and no loose jewelry
  • Pants and skirts must extend to the ankle with no rips
• Do's of Lab


  • Wash hands before and after lab
  • Wear proper lab attire
  • Disinfect the lab table at the start and end of each lab
  • Dispose of waste in designated areas
  • Place gloves in a biohazard waste container labeled "gloves only"
  • Cary test tubes containing media or cultures in a test tube rack
  • Notify the instructor if bacterial culture is spilled (and follow these steps)
• Steps to follow if bacterial culture is spilled

  1. Cover the spill with paper towel

  2. Saturate the paper towel with disinfectant and let it sit for at least 20 minutes

  3. Dispose of paper towels in appropriate biohazard waste container

  4. Disinfect the table following normal procedures
• Don'ts of Lab


  • Never have food, drinks, or cell phones visibly present
  • do not leave Bunsen burner unattended
  • do not lay tubes with culture down on the table
  • never pick up broken glassware with hands
• Proper handwashing technique


  1. Wet hands with warm water

  2. Lather hands by rubbing them together. (make sure to get the backs of your hands, between fingers, wrists, and under the nails)
• Steps of aseptic technique

  1. Hold the inoculating loop in the dominant hand, then pass the loop over the tip of the blue flame (loop should turn orange)

  2. All the loop cool down, which is about 10 seconds

  3. Remove the cap from the culture tube and hold the cap with the pinky finger. Pass the tube through the flame 3 times back and forth to prevent contamination.

  4. Insert loop into culture tube and remove a loopful

  5. Pass the lip of the culture tube over the flame 3 times back and forth and immediately recap it.

  6. Apply culture to medium (YOU KNOW THE REST!)
• Aseptic technique


  To prevent unwanted microorganisms from gaining access to our growth medium
• Pure culture


  A culture where all organisms are descendants of the same organism
• Colony


  A group of microorganisms that originate from a single cell and have grown into a distinct cluster
• Medium


  Anything in or on which we grow a microorganism
• Inoculum


  The portion of microorganisms taken from a culture and used to start a new culture on a new medium
• Quadrant streak


  Used to see bacterial colony isolation and morphology
• Zig-zag streak


  Used for isolation and is typically used if the sample is very diluted
• Types of Mediums


  • Agar slants are used to generate stocks of bacteria
  • agar deeps are used to grow bacteria that prefer an anaerobic environment
  • agar plants are used to isolate single colonies
  • broth cultures are used for growing large quantities of bacteria
• How to label a tube/plate

  Initials. Table #; organism, media, date
• E.g. of labeling

  • T.H. Table 1; E.coli, TSA, 9/11/2005
• Ocular lenses


  The eyepiece we view the specimen through
• Body tube


  Connects the ocular lenses to the nosepiece
• Arm


  Connects the body tube to the base and serves as a handle for carrying the microscope
• Rheostat


  Increases or decreases the light intensity
• Mechanical stage slide/drive knobs


  The 2 knobs below the stage will move the stage left and right (upper knob) and forward and back (lower knob)
• Fine adjustment knob


  The smaller inner knob that is used to precisely adjust the focus with all objectives
• Coarse adjustment knob


  The large outer knob that moves the stage up and down and is used for initial focusing under the scanning objective
• Base


  The foundation of the microscope
• Light source


  Small light located under the stage and is controlled by a light switch
• Condenser


  Concentrates the light before it reaches the specimen
• Iris diaphragm lever


  Controls the amount of light that reaches the specimen; provides greater contrast with the background
• Mechanical stage


  Frame with a stage clip to hold the slide containing the specimen
• Objective lenses


  4 lenses with different magnifying strengths
• Revolving nosepiece


  A rounded structure where the 4 objective lenses are attached to
• Procedure for oil immersion

  1. Obtain slide and be able to focus on the specimen under 40x objective

  2. Turn the objective lens until it is halfway between the high power (40x) and oil immersion (100x) objectives

  3. Place a drop of oil on slide ten completely rotating the lens, so the white oil immersion lens is in place

  4. Focus on the image using the fine adjustment knob

  5. After viewing the specimen, directly switch from the 100x objective to the 4x objective

  6. Remove the slide and clean everything
• Positive stain


  The dye is absorbed by the cells and makes them standout against the background (types of positive stains: crystal violet, methylene blue, and safranin)
• Negative stain


  The dye is absorbed by the background, outlining the uncolored cells (types of negative stains: nigrosine, eosin, and Indian ink)
• Procedure for gram staining of bacteria

  1. Prepare a heat-fixed smear

  2. Add a few drops of crystal violet to smear and let it sit for 60 seconds (crystal violet is the primary stain)

  3. Rinse the slide with distilled water (stopping at this step would be a simple stain)

  4. Add a few drops of gram's iodine to smear and let it sit for 60 seconds (gram's iodine forms a complex with crystal violet)

  5. Rinse the slide with distilled water

  6. Decolorize with 95% ethanol: let alcohol run over the surface of the slide until there is no more purple color

  7. Rinse the slide with water which stops the decolorizing process

  8. Add a few drops of safranin and let it sit for 60 seconds (counterstain)

  9. Rinse with water (shows if cells will be gram-positive or gram-negative)

  10. 10. Blot dry with bibulous paper
• Steps of negative staining


  Dragging nigrosine with another slide (DO NOT HEAT FIX)
• Psychrophiles


  0°C -15°C (very cold)
• Psychrotrophs


  4°C-25°C (cold)
• Mesophiles


  20°C – 45°C (temperate)