Microbiology and Laboratory Techniques: Bacteria, Stains, and Viral Pathogenesis

Total magnification

Objective × Ocular (10×)

Order of focusing

Start with 4× → coarse focus, Move to 10× → fine focus, Move to 40× → fine focus + adjust light, Add immersion oil, switch to 100× → fine focus ONLY

Iris diaphragm

Controls amount of light

Condenser

Controls focus of light

Bacterial morphology: Bacillus

Rods

Bacterial morphology: Coccus

Spheres

Bacterial morphology: Spirillum

Spiral

Aseptic Technique

Prevent contamination of You, The environment, The culture

Key rules of Aseptic Technique

Disinfect workspace before and after, No fans/open windows (minimize airflow), Wash hands before and after, Flame loop until red-hot before/after transfers, Flame tube mouths before/after opening

Transferring cultures: Slants

Streak from bottom to top

Transferring cultures: Broth

Transfer loopfuls or pipet 0.1 mL

Incubation temperature for most bacteria

25°C-37°C

Incubation temperature for fungi

Generally 25°C

Cationic stains

Positive stains such as methylene blue, crystal violet, safranin that bind negative cell parts (nucleic acids, wall)

Anionic stains

Negative stains such as Congo red, acid fuchsin that stain background or outer layers

Heat-fixing

Kills bacteria and sticks cells to slide

Simple Stain purpose

Show size, shape, arrangement

Gram-positive bacteria

Thick peptidoglycan, hold crystal violet, appear purple

Gram-negative bacteria

Thin peptidoglycan, lose crystal violet in ethanol, take safranin, appear pink

Acid-Fast bacteria

Retain carbol fuchsin, resistant to acid-alcohol, appear red

Non-acid-fast bacteria

Decolorized by acid-alcohol, counterstain methylene blue, appear blue

Endospores

Tough protein coat, hard to stain, only stain with heat + malachite green

Capsule Stain purpose

Detect capsules (protective glycocalyx)

Viable cell count

Only counts living cells using serial dilutions and plate counts

CFU/mL Formula

Example: 2 colonies on a plate, 0.1 mL plated, DF = 10³ → 2 / 0.1 × 10³ = 2 × 10⁴ CFU/mL

Disinfectants

used on surfaces; microbicidal or microbistatic.

Antibiotics

metabolic inhibitors, more specific, used medically.

Agar Disc Diffusion

Clear area around a disc = zone of inhibition (ZOI). Larger ZOI = more effective.

E. coli

Gram-negative rod

Bacillus subtilis

Gram-positive, spore-former

Penicillin

cell wall synthesis inhibitor

Streptomycin & tetracycline

disrupt ribosomes

Chloramphenicol, erythromycin

ribosome inhibitors

Gram Reaction Importance

Gram-negative (E. coli) are generally more resistant because of outer membrane.

Enterics

Gram-negative rods found in GI tract, includes E. coli, Salmonella, Citrobacter, Klebsiella, etc.

Kligler's Iron Agar (H₂S test)

Black precipitate = positive H₂S, No black = negative

Simmon's Citrate

Blue = positive (uses citrate), Green = negative

VP (Voges-Proskauer test)

Reagents: Barritt's A & B. Red/pink = positive (2,3-butanediol fermentation), No color change = negative

MR (Methyl Red test)

Must be read at day 5. Red = positive (mixed acid fermentation), Yellow/orange = negative

Phenol Red Tubes

Sugars tested: Dextrose, Lactose, Sucrose. Contains: pH indicator (phenol red), Durham tube (gas capture)

Results Interpretation

Yellow (acidic) = fermentation occurred, Bubbles in Durham tube = gas produced, Red/no change = no fermentation

Typical Organism Behaviors

E. coli: ferments lots of sugars, produces gas; B. subtilis: ferments dextrose + sucrose, not lactose; Micrococcus luteus: nonfermenter; Citrobacter freundii: strong fermenter; Serratia liquefaciens: variable, but can ferment several sugars

pGLO plasmid

contains: GFP (green fluorescing protein), bla gene (ampicillin resistance), araC + promoter (GFP only expressed with arabinose)

Key Steps of Transformation

CaCl₂ treatment → makes membrane permeable, Heat shock (ice → 42°C → ice) → DNA enters cell, LB recovery → cells begin expressing genes

Transformation Efficiency

You need: colonies on LB/amp/ara, DNA concentration (0.08 µg/µL), Volume used (10 µL), Fraction plated (100 µL / 510 µL)

Rabies Disease (Lyssavirus Rabies - RABV)

RABV is a negative-stranded RNA virus. It belongs to the Rhabdoviridae family, which are rod-shaped viruses.

Viral Proteins

The virus has 5 proteins: L, N, and P form the Ribonucleoprotein complex (RNP), supporting transcription, protection, and evasion.

Transmission & Habitat

It is most commonly transmitted through saliva, usually via bites.

Mechanism

The virus attaches to the host cell membrane and releases its RNP.

Stages & Symptoms

The disease follows distinct stages: Incubation (5 days to 2 years) is asymptomatic while the virus travels toward the CNS.

Acute Neurological Phase

Involves heavy damage to the brain and spinal cord, causing agitation, delirium, partial paralysis, foaming at the mouth, and hydrophobia.

Coma

Involves severe paralysis, irregular heart rate, and abrupt loss of heart function.

Rabies Treatment

Hard to treat because once symptoms appear, death is imminent.

Rabies Prevention

Relies on vaccinations (pets, humans, wildlife).

Post-exposure prophylaxis (PEP)

Involves administering Human rabies immune globulin and 4 rounds of vaccines.

Clostridium Difficile (C. diff)

An anaerobic, spore-forming, Gram positive bacteria.

C. diff Characteristics

Rod-shaped (bacilli) and often appears in chains or clusters. It is motile due to peritrichous flagella.

C. diff Toxins

Releases cytotoxins TcdA and TcdB.

C. diff Infection

Occurs via highly resistant spores that survive stomach acid.

C. diff Transmission

Through the fecal-oral route.

C. diff Mechanism of Disease

Antibiotic use disrupts the regular gut flora, allowing C. diff to proliferate unchallenged.

C. diff Symptoms

Includes diarrhea, inflammation of the colon (colitis), abdominal pain, and potentially toxic megacolon or sepsis.

C. diff Treatment

Includes antibiotics like Vancomycin and Fecal Microbiota Transplant (FMT) for recurring infections.

Human Papillomavirus (HPV)

A non-enveloped DNA virus belonging to the papillomaviridae family.

HPV Genome

Composed of double-stranded circular DNA.

HPV Transmission

Transmitted via skin-to-skin or sexual contact, and perinatal transmission.

HPV Life Cycle

Enters micro-abrasions, infects basal layer cells, and its DNA replicates with the host DNA.

HPV Disease Types

Low-risk types (e.g., HPV 6 and 11) cause genital warts; high-risk types (e.g., HPV 16 and 18) cause cervical and other cancers.

HPV Cancer Mechanism

Integrates its DNA into the host genome and produces oncoproteins E6 and E7, leading to uncontrolled cell division.

HPV Prevention

Largely preventable through vaccinations (Gardasil-9) and screenings (Pap smear, HPV DNA test).

Group A Streptococci (GAS)

A Gram + bacteria known as Streptococcus pyogenes.

GAS Characteristics

Cocci shaped and grows in chains, exhibiting β-hemolysis on blood agar.

Streptococcus pyogenes

It is cocci shaped and grows in chains. It exhibits β-hemolysis (complete destruction of red blood cells) on blood agar.

GAS Spread

GAS spreads through respiratory droplets and adheres to epithelial cells in the throat or skin, facilitated by surface proteins.

Streptolysin S

An extracellular toxin that causes red blood cell lysis and contributes to inflammation.

S-Protein

Acts as immune camouflage by binding lysed red blood cells to the bacteria surface, helping it survive detection in the bloodstream.

M-Protein

A fibrous protein that aids in immune evasion and prevents phagocytosis; it is coded by the emm gene.

F-Protein

Facilitates the initial steps of infection by binding to fibronectin in host membranes.

Strep Throat Symptoms

Symptoms include a sore throat that starts suddenly, inflamed throat and tonsils, fever/chills, and headache.

White Patches

Patients may have white patches or spots on the throat/tonsils or tiny red dots (petechiae) on the roof of the mouth.

GAS Treatment

GAS is treated with antibiotics like Penicillin and Amoxicillin, which inhibit the synthesis of the bacterial cell wall.

Prions

Prions are described as a 'Protein infection' (PrP^C) that is pathogenic without DNA.

Normal PrP^C Structure

Consists of 4 alpha helices.

Misfolded PrP^Sc Structure

Consists of 2 alpha helices and 2 beta sheets, creating amyloids.

Prion Transmission

Prion diseases are very rare (1 in 1,000,000) and can be acquired, inherited, or spontaneous.

Neurodegeneration Mechanism

The accumulation of PrP^Sc amyloids forms toxic aggregates that cannot be destroyed.

M1 Microglia

Release cytokines that weaken the blood-brain barrier, allowing neutrophils and leukocytes to cross and degrade neurons.

Prion Disease Symptoms

Symptoms include anxiety, insomnia, deterioration of cognitive function, muscle twitching, impaired coordination, and dementia.

Prion Disease Outcome

Prion diseases are always lethal, rapid (3 months to 3 years), and have no cure.

Measles Virus

Measles is a single-stranded negative-sense RNA Virus belonging to the Morbillivirus genus, in the Paramyxoviridae family.

Measles Viral Proteins

The virus has membrane glycoproteins; the H protein facilitates attachment to the host, and the F protein helps the viral envelope fuse with the host cell.

Measles Transmission

Measles is a highly contagious airborne virus transmitted through respiratory droplets or direct contact.

Measles Symptoms

Symptoms include cough, runny nose, sore throat, and fever, with a key diagnostic sign being Koplik Spots.

Koplik Spots

White spots with a red halo caused by immune destruction of cells in the mouth.

Measles Lethality Mechanisms

Measles can turn lethal through Immune Amnesia, Tissue Damage, and Neurological Issues.

Measles Vaccine

The Measles Vaccine was developed in 1963.

MMR Vaccine

Protects against measles, mumps, and rubella; developed in 1971.

Measles Elimination

Considered eliminated in the U.S. by the year 2000.

Leptospira

A bacterium and one of the most widespread zoonotic diseases.

Leptospira Characteristics

It is a spirochete—thin, flexible, and tightly coiled; highly motile using two periplasmic flagella.