Microbiology 5 I's, Media Types, and Staining Techniques

What are the 5 I's of microbiology and what does each involve?

Inoculation - introducing microbes into media to produce a culture

Incubation - providing proper growth conditions

Isolation - separating species into individual colonies

Inspection - observing microorganisms macroscopically and microscopically

Identification - determining the species using tests

Define culture, medium, sterile, colony, pure culture, mixed culture, and contaminated culture.

Culture: Growth of microorganisms in/on media

Medium (media): Nutrient-containing environment for growth

Sterile: Free of all life forms, including spores and viruses

Colony: Visible mass of microorganisms on solid media

Pure culture: One species only

Mixed culture: Two or more known species

Contaminated culture: Contains unwanted microorganisms

Why is sterile (aseptic) technique important?

revents contamination

Media and tools must be sterile

Prevents room air, hands, and other microbes from entering cultures

What is incubation?

Growth under proper conditions

Typical lab temperatures: 20-40°C

Incubators may control oxygen and carbon dioxide

Compare liquid, semisolid, and solid media.

Liquid: Broths; flow freely

Semisolid: Soft; used for motility testing

Solid: Contains agar; supports colony formation

What is agar?

Polysaccharide from red algae (Gelidium)

Solid at room temperature

Melts at 100°C and solidifies around 42°C

Compare defined and complex media.

Defined (synthetic): Exact chemical composition known

Complex: Contains ingredients such as blood, milk, yeast extract; exact composition unknown

Compare general-purpose and enriched media.

General-purpose: Grow many organisms (nutrient agar, TSA)

Enriched: Contain blood, serum, vitamins, or amino acids for fastidious microbes

What is selective media?

Inhibits some microbes while encouraging others.

What is differential media?

Allows multiple microbes to grow but shows visible differences.

What are examples of media that are both selective and differential?

MacConkey Agar

Mannitol Salt Agar

Why is blood agar important?

Enriched and differential medium

Detects hemolysis

Compare alpha, beta, and gamma hemolysis.

Alpha: Partial RBC destruction

Beta: Complete RBC destruction

Gamma: No hemolysis

What is a colony?

A visible mound of cells on solid media.

What is the streak plate method?

Uses an inoculating loop to thin out bacteria and obtain isolated colonies.

What are requirements for isolation?

Small number of cells

Solid agar surface

Petri dish

Inoculating loop

What methods are used for identification?

Macroscopic analysis

Microscopic analysis

Biochemical testing

Genotypic testing (DNA)

Immunologic testing (antibodies)

What can biochemical tests determine?

Nutrient requirements

Enzyme production

Metabolic products

Energy production methods

What are the three important properties of a microscope?

Magnification

Resolution

Contrast

What does the ocular lens do?

Eyepiece; closest to eye; forms visual image.

What does the objective lens do?

Closest to specimen; forms real image.

What does the condenser do?

Focuses light onto the specimen.

What are the magnifications of each objective lens?

Scanning = 4x

Low Power = 10x

High Dry = 40x

Oil Immersion = 100x

What is the ocular lens magnification?

Usually 10x

How do you calculate total magnification?

Ocular × Objective

Calculate the totals.

10 × 4 = 40x

10 × 10 = 100x

10 × 40 = 400x

10 × 100 = 1000x

What is the relationship between magnification and field of view?

As magnification increases, field of view decreases.

Trace the path of light through a microscope.

Light Source → Condenser → Specimen → Objective Lens → Ocular Lens → Eye

What is resolution?

Ability to distinguish two close objects as separate.

What is resolving power?

Minimum distance two objects can be apart and still appear separate.

What affects resolving power?

Objective lens quality

Wavelength of light

Why is oil used with the 100x objective?

Same optical properties as glass

Prevents light scattering

Increases numerical aperture

Improves resolution

What can oil immersion resolve?

Objects about 0.2 μm apart.

What is contrast?

Difference between an object and its background.

What determines contrast?

Refractive index (how much light bends).

What are the steps for preparing a smear?

Place sample on slide

Spread thin film

Air dry

Heat fix

Stain

Why is heat fixing important?

Kills bacteria

Secures cells to slide

Preserves cell structures

Prevents cells from washing away

Compare basic and acidic dyes.

Basic (cationic) dyes

Positive charge

Attracted to negatively charged bacterial cells

Stain cells

Acidic (anionic) dyes

Negative charge

Repelled by bacterial cells

Stain background

what is positive staining?

Basic dye stains bacterial cells.

What is negative staining?

Acidic dye stains the background around cells.

What is the simple stain procedure?

Prepare smear

Heat fix

Add basic dye (usually methylene blue) for 1 minute

Rinse

Observe

What information does a simple stain provide?

Presence or absence of bacteria

Shape of bacteria

What are the four Gram stain reagents and their functions?

Crystal Violet - primary stain

Gram's Iodine - mordant

Alcohol/Acetone - decolorizer

Safranin - counterstain

Compare Gram-positive and Gram-negative bacteria.

Gram Positive

Purple

Thick peptidoglycan

No outer membrane

Gram Negative

Pink/red

Thin peptidoglycan

Outer membrane present (phospholipids, lipoproteins, lipopolysaccharides)

What happens to Gram-positive bacteria during Gram staining?

Crystal violet enters

Iodine enters

CV-I complex forms

Thick peptidoglycan traps CV-I

Decolorizer cannot remove it

Cell remains purple

What happens to Gram-negative bacteria during Gram staining?

Crystal violet enters

Iodine enters

CV-I complex forms

Alcohol dissolves outer membrane

CV-I complex escapes

Cell becomes colorless

Safranin stains cell pink

What happens if you over-decolorize?

Gram-positive cells may appear Gram-negative (pink).

What happens if you under-decolorize?

Gram-negative cells may appear Gram-positive (purple).

Compare bacteria and eukaryotes.

bacteria

no nucleus, Dna Free in cytoplasm, no membrane bound organelles, cell wall contain peptidoglycan, unicellular

eukaryotes

nucleus present, DNA encolsed in nucleus, membran bound organelles, cell wall may be present or absent, uni or multicellular

What are the general characteristics of bacteria?

Kingdom Bacteria

Unicellular prokaryotes

Cell wall contains peptidoglycan

Reproduce by binary fission

Most are heterotrophs

Some are autotrophs

Some possess flagella

bacteria examples

Escherichia coli

Streptococcus

Staphylococcus

Bacillus anthracis

What are the three major bacterial shapes?

Coccus

Round

Spherical

Oval

Bacillus

Rod-shaped

Cylindrical

Club-shaped

Spirillum/Spirochete/Vibrio

Spiral or curved

What structures are found in eukaryotic cells?

Nucleus

Cytoplasmic membrane

Mitochondria

Endoplasmic reticulum

May also contain:

Cell wall

Chloroplasts

Flagella

How are eukaryotic flagella different from bacterial flagella?

10× thicker

More complex

Covered by cell membrane

9+2 microtubule arrangement

Move with whipping motion

What are the general characteristics of fungi?

Eukaryotic

Yeast (unicellular) or mold (multicellular)

Cell wall contains chitin

Heterotrophs

No motility structures

Reproduce by spores

Important fungal identification feature = spores

Define the following fungal terms:

yeast, hyphae, pseudohyphae, mycelium. septa, spore

Yeast: Unicellular fungus

Hyphae: Threadlike fungal cells

Pseudohyphae: Chain of yeast cells

Mycelium: Mass of intertwined hyphae

Septa: Cross walls within hyphae

Spore: Reproductive body

What is fungal dimorphism?

Ability of some fungi to exist as both mold and yeast.

Temperature-dependent:

25°C = Mold

37°C = Yeast

Usually seen in pathogenic fungi.

What are the 3 major types of mycoses?

systemic, subcutaneous, cutaneous

systemic

Systemic

Deep infections

Begin by inhalation

Start in lungs

Spread through body

subcutaneous

Subcutaneous

Enter through puncture wounds

Beneath skin

cutaneous

affect skin, hair, nails,

caused by dermatophytes

Rhizopus nigricans

Black bread mold

Opportunistic

Systemic

Mucor

Opportunistic

Systemic

Associated with mucormycosis

Aspergillus flavus

Produces aflatoxin

Liver cancer risk

Contaminated grain

Histoplasma capsulatum

Systemic mycosis

Bird droppings

Histoplasmosis

Cryptococcus neoformans

Pigeon droppings

Systemic mycosis

Fungal meningitis

Candida albicans

Normal microbiota

Opportunistic

Thrush

Vaginitis

Pneumocystis jirovecii

Opportunistic

PCP pneumonia

HIV/AIDS

Which fungi cause ringworm (tinea)?

A:

Trichophyton

Microsporum

Epidermophyton

Characteristics

Dermatophytes

Cutaneous mycoses

Digest keratin using keratinase

What are the general characteristics of algae?

Eukaryotic

Unicellular

Photoautotrophic

Chloroplasts present

Produce oxygen

Mostly aquatic

Cell wall contains cellulose

Sexual and asexual reproduction

What algae example must you know?

Pfiesteria

Dinoflagellate

Associated with human illness

Produces toxins

What are the general characteristics of protozoa?

Eukaryotic

Unicellular

Heterotrophs

No cell wall

Reproduce sexually or asexually

Classified by locomotion

Some form cysts

What are the four protozoan classifications?

Pseudopods

Amoeboid movement

Flagella

One or more flagella

Cilia

Numerous cilia

Apicomplexans (Sporozoa)

No adult locomotion structures

Intracellular parasites

Compare trophozoite and cyst.

A:

Trophozoite

Active

Feeding stage

Motile

Cyst

Dormant

Survival stage

Important in disease transmission

Entamoeba histolytica - disease, and transmission?

causes - amebic dysentery Symptoms:

Bloody mucus stool

Diarrhea

Fever

Weight loss

Transmission:

Fecally contaminated food/water, humans are primary hosts

Giradia lamblia - Classification, disease, and transmission?

Classification: Flagellate

Disease: Giardial enteritis

Symptoms:

Long-lasting diarrhea

Abdominal pain

Greasy, foul-smelling stools

Transmission:

Contaminated water

Trichomonas vaginalis - Classification, disease, and transmission?

Classification: Flagellate

Disease:

STI

Vaginitis

Female symptoms:

White-green frothy discharge

Transmission:

Sexual contact

Trypanosoma - Classification and diseases?

Classification: Flagellate

Chagas Disease

Triatoma (kissing bug)

African Sleeping Sickness

Tsetse fly

Balantidium coli - Classification and disease?

Classification: Ciliate

Disease:

Balantidial dysentery

Transmission:

Contaminated water

Toxoplasma gondii - Classification, hosts, disease, and transmission, risks?

Classification: Apicomplexan

Disease: Toxoplasmosis

Definitive Host: Cats

Intermediate Hosts: Humans and other animals

Transmission:

Cat feces

Undercooked meat

risk:

Liver/spleen enlargement

Liver failure

Pregnancy Risks:

Blindness

Hydrocephalus

Neurological damage

Stillbirth

33% fetal transmission rate

Plasmodium - Classification and disease?

Classification: Apicomplexan

Disease: Malaria

Symptoms:

Chills

Fever

Sweating

Fatigue

Nausea

Species:

P. falciparum

P. vivax

P. malariae

P. ovale

What are the definitive and intermediate hosts of Plasmodium?

Definitive Host

Female Anopheles mosquito

(Contains sexual stage)

Intermediate Host

Human

(Contains asexual stage)

Describe the asexual portion of the malaria life cycle.

Mosquito injects sporozoites

Sporozoites travel to liver

Schizogony occurs

Thousands of merozoites produced

Merozoites infect RBCs

Become trophozoites (ring stage)

RBC bursts

More merozoites released

Chills and fever occur

Cycle repeats

Plasmodium

Malaria symptoms occur every 48-72 hours

200 million cases/year

90% in Africa

500,000-1 million deaths/year

Ring stage terminology

Schizogony terminology

What are the general properties of viruses?

Acellular (not cells)

Obligate intracellular parasites

Infect bacteria, fungi, algae, protozoa, plants, and animals

Cannot reproduce independently

Use host cell machinery to reproduce

Best described as active or inactive rather than alive or dead

May influence evolution and host genetics

Part of the normal human microbiome

Why are viruses considered nonliving?

Cannot carry out metabolism independently

Cannot reproduce on their own

Must invade a host cell to multiply

Lack protein synthesizing machinery

Are inactive outside a host cell

How large are viruses?

Smaller than most bacteria

Parvovirus ≈ 0.02 μm

Some giant viruses (Pandoravirus) can be as large as bacteria

Require electron microscopes for visualization

What are the basic components of a virus?

Nucleic acid core

Capsid (protein coat)

Some viruses also contain:

Envelope

Spikes

Enzymes

Define capsid, nucleocapsid, envelope, spikes, and virion.

Capsid = protein shell surrounding nucleic acid

Nucleocapsid = capsid + nucleic acid

Envelope = modified host membrane surrounding some viruses

Naked virus = virus lacking envelope

Spikes = attachment proteins used to bind host cells

Virion = complete infectious virus particle

What type of genetic material do viruses contain?

DNA OR RNA

Never both

Genome contains only genes needed to invade and control host cells

Examples:

Hepatitis B = 4 genes

Pandoravirus = 2,500+ genes

How are viruses classified?

Informally:

Animal viruses

Plant viruses

Bacterial viruses

Enveloped or naked

DNA or RNA

Helical or icosahedral

Formal classification:

Structure

Chemical composition

Genetic makeup

What are the six phases of the animal virus life cycle?

Adsorption

Penetration

Uncoating

Synthesis

Assembly

Release

What is adsorption?

Virus attaches to specific receptors on host cells.

First step of infection.

What is host range?

The types of cells a virus can infect.

Examples:

Hepatitis B → liver cells only

Poliovirus → intestinal and nerve cells

Rabies virus → many mammalian cells

What are penetration and uncoating?

Penetration:

Virus enters host cell

Often by endocytosis

Uncoating:

Capsid and envelope removed

Viral nucleic acid released

What occurs during synthesis?

Viral nucleic acid takes over host machinery

New viral genomes produced

New viral proteins produced

Remember:

RNA viruses replicate in cytoplasm

DNA viruses replicate in nucleus

What occurs during assembly and release?

Assembly:

Viral parts assembled into new virions

Release:

Viruses leave cell by budding or exocytosis

Cell may survive temporarily

How can viruses cause cancer?

Oncogenic viruses may:

Carry cancer-causing genes

Produce proteins that disrupt growth regulation

Approximately 13% of human cancers are linked to viruses.

What are bacteriophages?

Viruses that infect bacteria

Discovered in 1915

Every bacterial species has at least one bacteriophage

Can increase bacterial pathogenicity

Examples:

T2

T4

What is the lytic cycle?

Life cycle that ends with destruction of the bacterial cell.

Steps:

Attachment

Penetration

Synthesis

Assembly

Lysis and release

Result:

Host cell dies