Micro Lab Test 1

Biohazard Can

All items that contact bacteria

Sharps Container

Slides, swabs, pipettes

Discard Cart

Glassware

Trash

Gum, paper, personal items

First Aid Kit

Next to right front sink

Eyewash Station

Front and Back Sinks

BSL-1

No disease

No PPE

Handwash

BSL-2

Indigenous, contact/splash, Lab Coat and gloves, Restricted access

BSL-3

Indigenous or exotic, serious/lethal, aerosol, PPE + respirator, negative airflow,

BSL-4

Exotic, no treatment, full body suit, clothing change.

Ubiquity of Microorganisms:

Microbes are virtually everywhere

Sterile Exceptions:

Blood and cerebrospinal fluid (CSF) in the human body; clean rooms; sterilized culture media.

Agar Origins

• Seaweed extract

• Melt: 85 c

• Solidifies: 25 c

• solid up to 60 c

Gelatin Shortfalls

melts at human body temperature (37 °C), bacteria digest it

Agar Advantages

Thermally stable across incubation ranges and resistant to microbial degradation.

Robert Koch

Germ theory of disease; designed initial glass dish for culture.

Angelina Fanny Hesse

Suggested using agar instead of gelatin to her husband, Walther Hesse, in Koch's lab.

Julius Petri

• Modified Koch’s glass plate to invent the modern Petri dish.

BSL-1 Example

Bacillus subtilis

BSL-2 Example

Staphylococcus aureus

BSL-3 Example

Mycobacterium tuberculosis

BSL-4 Example

Ebola virus

Broth Innoculation

Insert loop and twirl/twist without touching tube walls.

Slant Innoculation

Draw loop up the surface in a zig-zag pattern from bottom to top.

Plate to tube Innoculation

Lift plate bottom off lid (leave lid on bench to shield dust), touch loop straight down, and lift up (do not drag).

Brightfield

• Light passes/reflects

• Simple absorption/transmission

• Poor internal structure

• Staining required

Percent water of cells

80%

Phase Contrast

• Refractive index variation

• Some internal, some external

• Difference in refractive index to light sensitivity

Darkfield

• Dark background

• Scattering of light

• External only

Fluorescence

• High-intensity light

• Fluorophore photon emission

• Can see internal structures

• staining required

Total Magnification

Ocular Lens Power (10x) x Objective Lens Power

Lens magnification

• Oculars = 10x

• Low power = 10x

• High dry = 40x

• oil immersion = 100x

immersion Oil (100x ONLY)

Matches the refractive index of glass, reducing light refraction and increasing resolving power.

Parfocal

Image remains in approximate focus when changing objectives.

1. Primary stain

Crystal Violet

Crystal Violet

Stains all cells purple by penetrating walls

2. Mordant

Gram’s Iodine

Gram’s Iodine

Forms CV-I complex, Plugs pores in the peptidoglycan (Gram +)

Decolorizer

95 % Ethanol

Ethanol

Washes CV-I out of this GRAM - walls (colorless) and shrinks Gram + walls

Counterstain

Sefranin

Sefranin

Stains decolorized Gram - Cells (PINK/RED)

Gram Positive Cocci

• Purple

• Pairs, tetrads, chains, clusters

Gram Negative Cocci

• Pink/Red

• Pairs or Tetrads

Rods (Bacilli)

• Gram-positive

• Purple

• Single rods, chains

Gram Negative Straight Rods

• Pink/Red

• Single rods

Curved or Spiral Rods

• Gram-negative

• Pink/Red

• Single curved or spiral

Catalase

• Tests for an enzyme that breaks down hydrogen peroxide

• Bubbles = Positive

• No bubbles = negative

• 2H₂O₂→ 2H₂O + O₂

• Reagent: 3% hydrogen peroxide

Catalase Organism Association

• Staphylococcus (+)

• Streptococcus/Enterococcus (-)

Oxidase

• Test Cytochrome C Oxidase Enzyme

  • in bacteria, electron transport chain

• Blue/Purple = Positive

• (+) Pseudomonas and bacillus cereus

KOH (Potassium Hydroxide)

• Identifies Gram-negative

• Lysis of this wall-less Gram-negative bacterium

• Releases DNA (viscous strings)

Selective Media

• Allows only certain microorganisms to grow while inhibiting the growth of others

MSA (Mannitol Salt Agar)

• Selective and Differential Media

• 7.5% Salt

• Differentiates mannitol fermentation

• Positive: Growth + Yellow Agar (Acid +)

• Negative: Red Agar, Colorless, No growth

• Contains mannitol and a pH indicator (phenol Red)

MSA

• Specific for staphylococcus

• Reagent: 7.5% Salt

• Indicator Phenol Red

Differential Media

• Causes some bacteria to take on an appearance that distinguishes them

pH Indicating Molecules

• Phenol Red

• bromothymol blue

• methyl Red

Fermentation of Carbs

Produces acidic products

Phenol Red

• Neutral = Red

• Acidic = yellow

Starch Hydrolysis

• Amylase production (flood with Gram's Iodine)

• Differentiates based on the ability to hydrolyze starch

• Positive: Clear zone around streak

• Negative: Dark Agar

• Bacillus (+)

Phenol Red Carbohydrate Tubes

• Differentiates based on carb fermentation

• Positive: Yellow

• Negative; Red

• Incubate for 48 hrs

Sheep’s Blood Agar (5%)

• Differentiate hemolytic activity

• Gamma = no discoloration

• Beta = Clearing of the media

• Alpha = Yellowing of the media

• Streptococcus, Enterococcus, Bacillus

Bile Esculin Slant

• Ferric Citrate

• Negative = No color change

• Positive = Black

• (+) for Enterococcus faecalis

BHI broth with 6.5% Salt

• High Salt

• Turbid = Positive

• Key foe enterococcus

Coagulase

• Rabbit Plasma

• solidified fibrin clot

• Specific to staphylococcus Aureus

Obligate Aerobe

• Require O2

• Aerobic Respiration

• Grows at the top

• (+) Catalase

• (+) SOD

Facultative Aerobe

• Prefers O2 but can ferment

• Catalase (+)

• SOD(+)

Aerotolerant Anaerobe

• Ignores O2, only ferments

• Catalase (-)

• SOD (+)

Obligate Anaerobe

• O2 is toxic

• Catalase (-)

• SOD (-)

• Anaerobic respiration or fermentation

Gram Stain is

Differential Stain

Peptidoglycan is in

Cell wall

NItrate Reduction: Anaerobic

1. Step 1. Reagent A & Reagent B are added

   1. Turns Red = Incomplete Positive (Reduced NO3 to NO2)

   2. Stays Clear = inconclusive → step 2

      1. Add Zinc Dust

         1. Stays Clear = Complete Positive (Reduced NO3 to NH3 or N2)

         2. Turns Red = Negative ( forced to nitrate by zinc)

aerobic respiration

bacteria utilize oxygen (O₂) as the terminal electron acceptor

Strict aerobes and facultative anaerobes typically produce

Catalase

Strict anaerobes lack both

catalase and peroxidases,

superoxide dismutase (SOD),

converts superoxide iding oxygen tolerance;

What are reactive oxygen species (ROS)?

armful oxygen byproducts formed during aerobic respiration, including superoxide (O₂⁻) and hydrogen peroxide (H₂O₂).

Which bacteria are usually catalase positive?

Strict aerobes and facultative anaerobes.

Why are strict anaerobes sensitive to oxygen?

They lack catalase and peroxidases.

What does superoxide dismutase (SOD) do?

Converts superoxide radicals into H₂O₂ and O₂.

Final product of cytochrome c oxidase activity?

Water

What enzyme hydrolyzes starch?

Amylase, Starch molecules are too large to enter cells.

What makes BEA selective?

Bile salts (oxgall).

Which bacteria should be tested with coagulase?

Gram-positive staphylococcus-like bacteria.