Lab Midterm

1.  Introduction to microbiology lab and lab safety

2. Introduction to light microscope

3. Simple microscope- eyepiece, nose piece, stage, body, light, & base

4. Names of the lenses

   1. 4x= scanning lens

   2. 10x= low power objective lens

   3. 40x= high power objective lens

   4. 100x= oil immersion lens

5. Ubiquity of microorganism

6. 4.      Simple staining: Uses a single dye to visualize morphology

   1. Reagent: Methylene blue, crystal violet, or safranin

   2. Steps:

      1. Prepare a bacterial smear on a slide and heat-fix.

      2. Apply the stain for 30-60 seconds.

      3. Rinse with water and blot dry.

      4. Observe under the microscope.

7.   Wet mount preparation:

   1. Reagent: Saline or water

   2. Steps:

      1. Place a drop of liquid sample on a clean slide.

      2. Cover with a coverslip.

      3. Observe under the microscope using low and high power.

8. Gram stain

   1. Components: a differential staining technique that distinguishes bacteria based on cell wall composition.

   2. Characteristics: differentiates gram-positive (thick peptidoglycan) vs gram-negative (thin peptidoglycan)

   3. Positive vs Negative

      1. Gram-positive: Purple

      2. Gram-negative: Pink

   4. Reagents & order of use:

      1. Crystal violet (1 min; primary stain)

      2. Iodine (1 min; mordant)

      3. Alcohol (15 secs; decolorizer)

      4. Safranin (1 min; counter stain)

   5. Steps:

      1. Prepare and heat-fix a bacterial smear.

      2. Apply crystal violet for 60 sec, rinse.

      3. Apply iodine for 60 sec, rinse.

      4. Decolorize with alcohol for 10-15 sec, rinse.

      5. Counterstain with safranin for 30 sec, rinse.

      6. Blot dry and observe.

   6. Representative Organisms & Results:

      1. Gram-positive: S. aureus (purple)

      2. Gram-negative: E. coli (pink)

   7. Results for E. coli & S. aureus:

      1. E. coli: Gram-negative (pink)

      2. S. aureus: Gram-positive (purple)

9. Negative stain

   1. Components: stains background, not cells

   2. Characteristics: no heat fixing; preserves cell shape & size

   3. Positive vs Negative Results: bacteria appear unstained on a dark background

   4. Reagent: Nigrosin or India ink (air dry)

   5. Steps:

      1. Place a drop of stain on a slide.

      2. Mix bacteria with stain and use another slide to spread.

      3. Air dry and observe.

   6. Repsentative Organism & results: used for spirochetes & capsules

   7. Results for E. coli & S. aureus:

      1. E. coli: Unstained rods on dark background

      2. S. aureus: Unstained cocci on dark background

10. Capsule stain

    1. Components: stains capsule & background; capsule remains clear

    2. Characteristics: detects capsules (virulence factor)

    3. Positive vs Negative Results

       1. Positive: clear halo around cells

       2. Negative: no clear halo

    4. Reagents: Congo red or India ink, counterstain (crystal violet) (Air dry)

    5. Steps:

       1. Mix bacteria with primary stain.

       2. Spread across the slide and air dry.

       3. Counterstain and rinse.

       4. Observe under oil immersion.

    6. Representative Organisms & results:

       1. Klebsiella pneumoniae – Capsule positive

    7. Results for E. coli & S. aureus:

       1. E. coli: Can produce capsule (depends on strain)

       2. S. aureus: Usually capsule negative

11. Endospore stain

    1. Components: Identifies bacterial endospores

    2. Characteristics: endospores resist heat, chemicals

    3. Positive vs Negative Results:

       1. Positive: Green endospores inside red cells

       2. Negative: No green spores

    4. Reagents:

       1. Malachite green + steam (5-7 mins)- stains spores

       2. Water rinse- removes stain from cells

       3. Safranin (1 min)- counterstains cells

    5. Steps:

       1. Prepare and heat-fix a bacterial smear.

       2. Apply malachite green and steam for 5 min.

       3. Rinse with water.

       4. Counterstain with safranin for 30 sec.

       5. Blot dry and observe.

    6. Representative Organisms & results:

       1. Bacillus cereus – Endospore positive

    7. Results for E. coli & S. aureus:

       1. E. coli: No spores

       2. S. aureus: No spores

12. Acid fast stain

    1. Components: Identifies Mycobacterium with waxy cell walls

    2. Characteristics: Acid-fast bacteria retain carbol fuchsin after decolorization

    3. Positive vs Negative results:

       1. Positive: Red (acid-fast)

       2. Negative: Blue

    4. Reagents:

       1. Carbol fuchsin + steam (5 mins)- primary stain

       2. Acid-alcohol (10 sec)- decolorizer

       3. Methylene blue (1 min)- counterstain

    5. Steps:

       1. Prepare and heat-fix a bacterial smear.

       2. Apply carbol fuchsin and steam for 5 min.

       3. Rinse and decolorize with acid-alcohol.

       4. Counterstain with methylene blue for 30 sec.

       5. Blot dry and observe.

    6. Representative Organisms & Results:

       1. Mycobacterium tuberculosis – Positive (red)

    7. Results for E. coli & S. aureus:

       1. E. coli: Negative (blue)

       2. S. aureus: Negative (blue)

Introduction to media:

1. Tryptic soy agar/ broth:

   1. Selective/ Differential: general purpose

   2. Results for E. Coli & S. aureus:  growth observed for both

2. Selective and differential medias:

   1. Mannitol salt agar

      1. Selective for: Staphylococcus (high salt)

      2. Differential for: Mannitol fermentation

      3. Positive vs Negative Results:

         1. Positive (fermenter): Yellow (S. aureus)

         2. Negative: Pink/red

      4. Results for E. coli & S. aureus:

         1. E. coli: No growth

         2. S. aureus: Yellow

   2. MacConkey agar

      1. Selective for: Gram-negative

      2. Differential for: Lactose fermentation

      3. Positive vs Negative Results:

         1. Positive (fermenter): Pink (E. coli)

         2. Negative: Colorless

      4. Results for E. coli & S. aureus:

         1. E. coli: Pink

         2. S. aureus: No growth

   3. Eosin Methylene Blue agar

      1. Selective for: Gram-negative

      2. Differential for: Strong lactose fermenters

      3. Positive vs Negative Results:

         1. Positive: Green metallic sheen (E. coli)

         2. Negative: Colorless

      4. Results for E. coli & S. aureus:

         1. E. coli: Green sheen

         2. S. aureus: No growth

   4. Xylose lysine deoxycholate agar

      1. *Selective/Differential

         1. Identification of Salmonella & Shisella

         2. Sodium deoxycholate (kills gram positive)

         3. Xylase, Lysine, ferric Ammonium citrate à differential

         4. Phindicator => phenol Red

         5. Acidic => yellow

         6. Basic => red

         7. Shisella; use lysine à Red colonies

         8. Salmonella: ferment xylase & then lysine à Red w/ black center

         9. E. Coli => ferment sucrose, lactose => yellow

   5. Fluid thioglycollate broth

      1. Used to check O₂ sensitivity of bacteria

      2. Sodium thioglycolate consume O₂ from media

      3. Resazurin (redocindicator) pink in xce of O₂

      4. Bacteria grow in different places based on their O₂ tolerance

3. Utilization media:

   1. DNase agar

      1. Bacteria used

         1. B. cereus

         2. S. aureus

         3. S. epidemidis

         4. E. coli

      2. Check organism’s ability to produce enzyme

      3. DNase break down DNA

      4. DNA w/ toludine bluse

         1. Gram positive => blue => pink

         2. Gram negative=> blue => blue

   2. Starch agar

      1. Ability to produce alpha- amylase enzyme

      2. Add iodine agter incubation to see activity

      3. Gram positiveà starch break down around colony

      4. Clear when I₂ added

      5. Gram negative à No break down of starch, I₂ is retained

      6. Blusish/orange color

4. Enriched agar:

   1. Blood agar:

      1. Selective/Differential: Enriched, differentiates hemolysis

         1. Results for E. coli and S. aureus:

            1. E. coli: Beta-hemolysis (sometimes)

            2. S. aureus: Beta-hemolysis

   2. Chocolate agar

      1. Components:

         1. Enriched medium containing lysed red blood cells

         2. Provides NAD (V factor) and Hemin (X factor) for fastidious bacteria

      2. Characteristics:

         1. Supports growth of fastidious organisms that do not grow on regular blood agar

      3. Positive vs Negative Results:

         1. Positive: Growth of fastidious bacteria like Neisseria and Haemophilus

         2. Negative: No growth (organism does not require enriched media)

      4. Reagents & Order of Use:

         1. Pre-prepared enriched agar

         2. Inoculate bacteria and incubate at 35-37°C with 5-10% CO₂

         3. Observe for bacterial growth

      5. Representative Organisms & Results:

         1. Neisseria meningitidis – Positive (growth)

         2. Haemophilus influenzae – Positive (growth)

      6. Results for E. coli & S. aureus:

         1. E. coli: Can grow, but does not require Chocolate Agar

         2. S. aureus: Can grow, but does not require Chocolate Agar