Lab Final

Total Magnification

Product of the magnification of the ocular (10x) and the objective lens

Ocular x Objective

10 × 4 = 40x (LP)

10 × 10 = 100x (LP)

10×40 = 400x (HP)

10 × 100 = 1000x (OI) - Oil immersion

Working distance

The distance from the end of the objective lens to the top of the slide when the specimen is in focus

Higher magnification =

shorter working distance​

Resolving Power

Ability of a microscope to differentiate two closely spaced objects as being distinct from one another​

Resolving power =

Wavelength of light (λ)/2xN.A. = 500 nm/2x1.25 = 200 nm or 0.2 um​

wavelength of light = 500 nm!!

Resolving power increases as

magnification of the objective lens increases​

Shorter wavelength of light

increases resolving power​

Locations of Bacteria​

• Non-living surfaces (fomites, phone, table top)

• Living Surfaces​

• Dust particles in the air​

Majority of microorganisms

are nonpathogenic and don’t cause disease

Food-borne Infections

• Hepatitis A​

• Salmonella

Water borne Infections - its an answer on the final

Cholera

Nutrient broth/agar

• Peptone

• Beef extract

• Distilled water

• Agar

Positive growth

broth = cloudy

slant = agar plate, see bacteria growing

agar = cloudy at the stable line

Sterilization

• Autoclave or steam sterilization uses steam under pressure. Sterilization of culture media, reagents, and laboratory tools​

• Dry Heat, Uses an oven to generate hot, dry air, Sterilization of glassware and moisture-sensitive preparations​

• Filter sterilization​Uses a membrane to trap microorganisms. Sterilization of heat sensitive liquids​

• Flame sterilization​, Uses an open flame to incinerate microorganisms​, Sterilization of tools like inoculating loops/needle

Pure Culture, Colony, Pure Colony

Pure culture: contains only a single type of organism (e.g. thebacterium E. coli). This can also exist in a broth culture!

Colony is a group of bacteria growing in/on an agar medium​

Pure colony arises from a single bacterium through cell division​

MacConkey Agar

Bile salt, inhibit gram +, and help gram -

Gram - that ferment lactose to acids cause the indicator to change color to red, so that red or pink colonies appear

E. Coli will turn red

• Peptone​

• Proteose peptone​

• Lactose​

• Bile salts No. 3​

• NaCl​

• Agar​

• Neutral red​

• Crystal violet

B. Megaterium

will grow black in bile-esculin agar

Basic and Acidic Dyes

A basic dye consists of a colored group and a negative ion(Dye+Cl-)​

An acidic dye consists of a negatively charged color group and a positive ion (Na+Dye-)

Crystal violet (purple), safranin (pink or red), methylene blue(blue), and carbolfuchsin (pink)

We use basic dyes to stick

Preparation of smears from liquid and solid media

Hold the slide with a clothespin

a small drop of water for the solid colony is best (or you’ll be waiting a long time for it to dry)​

a small dab of bacteria from the colony is best (or you will have too many bacteria on the slide)​

Simple stain

everything will be pink

Gram stain - postiive or negative

Capsules and Virulence

Capsules enhance virulence (the severity of a disease)

• Interfere with phagocytosis by macrophages​

• Promote infection by adhering to host cells​

• Gram-negative encapsulated bacteria are more resistant to the lethal action of complement (causing lysis of cells) and other serum factors​

• Enhance resistance to the lethal actions of heavy metals, dehydration, and attack of bacterial viruses​

Klebsiella pneumoniae

has a Capsule

Negative stain procedure

• A small sample of a bacterial culture is mixed with India inkand spread over the surface ofa slide, then heat fixed​

• The bacterial film is stained with crystal violet for 1 minute, rinsed, and blotted dry​

• Cells stain violet and capsules appear as clear bright halos around the stained cells​

• The background is stained dark gray or black by the ink

Quellung Test

Valuable laboratory test forthe sero-identification ofspecific encapsulatedbacteria

The Gram Stain in Diagnosis of Disease

Identify different pathogens, lots of different body things

• spinal fluid (meningitis)​

• urethral exudate (gonorrhea)​

• urine sample (urinary tract infections) ​

Gram + and - stain

+purple

-purple, decolorize, clear, and then pink with safranin

Gram + cell wall

thick ppptidoglican, plasma membrane

Gram - cell wall

Phosphlipide bilayer, thin peptidoglycan, plasma membrane

Endospores

Under pressure turn into endospore

inactive state to survive harsh conditions

Bacillus & Clostridium

Endospore Stain

• Components​, Primary stain (malachitegreen)​, Decolorizer (water)​, Counterstain (safranin)​

• Procedure​, A smear is flooded withmalachite green and the stainis forced into the spore wallby steam​, After removal of excess stainwith water a counterstain(safranin) is applied​, Stained spores appear greenand the sporangium isstained pink

Two chemicals responsible for endospore resistance

• Calcium ​

• Dipicolinic acid​

The Acid-Fast Stain

RETAIN DYE ARE ACID FAST

NON ACID FAST RETAIN BLUE WITH METHYLENE BLUE

Mycobacteria

Lipid covering called mycolic acid

Mycobacteria frequently do not stain uniformly with the Gram-stain —> Acid-fast stain (Variable Results) ​

What dye does mycolic acid typically bind?

Carbolfuchsin with heat.

Flagella

Flagellum

True motility

Brownian movement

True Motility - Directed motion of bacteria, using energy

Brownian movement - random jiggling motion

Motility demonstration 3

Flagella stain, Hanging drop preparation (living), culture ( Stab inoculation of bacteria into motility agar medium in tubes)

Flagella arragements

Atrichouce - none

Mono - one

Amphitritchouse - 2 on each end

Lophotrichose - cluster on one end

Peritrichous - hair all over

Psychrophiles, Mesophiles, Thermophiles

Psychophiles -5 - 20 C

Mesophiles 20-45 C MOST DISEASE-CAUSING PATHOGENS

Thermophiles 45 - 60 °C

• Facultative thermophiles will grow at 37℃, but grow best at higher temperatures

• Obligate thermophiles will only grow at elevated temperatures

No cell wall

Isotonic - leave and enter same rate cell stays same

Hypotonic - Burst

Hypertonic - Shrink

Cell Wall (BACTERIA)

Isotonic - stays the same

Hypotonic - water goes in, cell wall keeps it from burst

Hypertonic - water rush out, cell wall stays safe, cell membrane shrinks PLASMOLYSIS!!

Extreme Halophiles

Require high salt concentrations for survival

• Stabilize the cell wall and enzymes

• K⁺ ions to survive in high salt environments

• Water then enters the cells to prevent dehydration, extremely saline environments in which they grow

Stomach

Many enteric pathogens are protected by food particles as they pass thru the stomach  and aren’t destroyed by stomach acid

Mouth

Formation of dental caries depends on the production of acid by streptococci and lactobacilli

Acid demineralizes tooth enamel and promotes caries formation

The pH at the tooth surface can be reduced to 5 or less

ACID PROMOTES CAVIEIS

pH and inhibitaiton

Very low and high pH interferes with enzyme activites, denature proteins, and can block transport of substance into cells

pH environments

• Neutrophiles

  • Most microorganisms grow well in a pH range of 6 to 9 and are termed neutrophiles

  • Fungi can tolerate a wider range of pH than bacteria since they grow well in a pH range of 4 to 9

• Obligate acidophiles

  • Only grow at the low pH of 2 to 3

  • When these cells are grown in a neutral pH environment, the cell membrane lyses and the cells die

  • Hydrogen ions are necessary to stabilize the cell membranes of these cells

• Alkalophiles

  • Grow at a pH range of 10 to 12

Transient / Resident Flora

• Transient flora

  • Bacteria (and other microbes) that are commonly found on the skin

• Resident flora

  • Bacteria (and other microbes) that are more permanent

  • Not easily removed by washing

  • Examples: Diphtheroids such as Corynebacterium and Propionibacterium, and staphylococci such as S. epidermidis

Hand washing does not remove resident, but it removes transient flora

longer than 2 minutes, it will take some of the resident flora out

Not all but some

Ignaz Semmelweis

hand washing

Glycolysis

Net gain of 2 ATP molecules, 2 molecules of NADH, and 2 molecules of pyruvic acid

Lactose + H2O (Lactase)—> D-galactose + D-glucose

Non-fermenter

Include aerobic, non-spore-forming bacilli that are non-saccharolytic and do not use or metabolize carbohydrates in a strictly oxidizing fashion

• Non-saccharolytic

Homolactic & Heterolactic Fermentation

Homolactic

• Some streptococci and lactobacilli produce primarily lactic acid from pyruvic acid

Heterolactic

• Other streptococci and lactobacilli produce lactic acid, ethanol, and carbon dioxide

Thermophilic organisms (surivive pasterization)

Streptococcus (S. lactis) and Lactobacillus

• Some spore formers in the genus Bacillus

Organisms found in raw milk

• Common genera:

  • Bacillus

  • Streptococcus

  • Lactobacillus

• Pathogens:

  • Mycobacterium tuberculosis (TB)

  • Brucella (brucellosis)

  • Salmonella typhi (typhoid fever)

  • Coliforms (E. coli)

  • Coxiella burnetii (Q fever)

  • Listeria monocytogenes (Listeriosis)

Raw Milk Standards

100,000 bacteria/mL - raw

20,000 bacteria - grade A pasteurized

Bacteriostatic/Bactericidal Agents

• Bacteriostatic agents

  • Stop the multiplication of bacteria, but do not necessarily kill them

• Bactericidal agents

  • Kill bacteria

Categories of Chemical Antimicrobial Agents

• Phenols

• Halogens

• Alcohols

• Quaternary Ammonium Compounds (Quats)

Halogens

Only ones that are oxidizing agents

Antibiotic

• Penicillins – cell wall

• Cephalosporins – cell wall

• Tetracyclines – inhibit protein synthesis

• Macrolides – inhibit protein synthesis

• Aminoglycosides – inhibit protein synthesis

• Quinolones – interfere with DNA replication

• Polymyxins – damage cell membrane

Broad/Narrow Spectrum Antibiotics

Broad spectrum antibiotics

• Effective against Gram + and Gram – bacteria

• Kills wide range of disease causing bacteria 

Narrow spectrum antibiotics

• Effective against specific bacteria

Wavelength

100 - 400 nm

most effective = 260 nm

Formation of Pyrimidine Dimers

When two pyrimidine bases are next to each other on the same DNA strand

UV radiation in the range of 260 nm is strongly absorbed by purine and pyrimidine bases in DNA

Photoreactivation

eliminate damage produced by UV light (fixes dimers)

photolyase enzyme

In the presence of longer wavelength light (e.g., 500 nm)

S. aureues and skin infections

Boils, carbuncles, impetigo, toxic shock syndrome and staphylococcal scalded skin syndrome (SSSS)

TSA

any bacteria, no difficination

MSA

selective medium, for halotolerant organisms like the staphylococci

Which medium is greatest variety of colony types?

TSA can grow more

MSA can differeiencant more

Only happens on a blood agar

Hemolysis

Gamma - None

Beta - Complete

Alpha - Partial

Differentiation of Staphylococcal Cultures

• Staphylococcus aureus

  • Catalase pos.*

  • Coagulase pos.

  • Mannitol pos. (fermentation)

• Staphylococcus epidermidis (and other staph.)

  • Catalase pos.

  • Coagulase neg.

  • Mannitol neg.

*Staphylococci are catalase pos., Streptococci are catalase neg.

Bacterial Respiratory Infections

Strep throat”/ dental caries

• Streptococcus pyogenes/ S. mitis, S. mutans

Abscesses

• Staphylococcus aureus, S. epidermidis

Otitis media

• Haemophilus influenzae

Sinusitis, otitis media, pneumonia

• Moraxella (Branhamella) catarrhalis

Streptococcal pharyngitis
“Strep Throat”

Causative agent

• Streptococcus pyogenes (Group A, beta-hemolytic, gram-positive)

Streptococcus Mutans

Dextran surrounds the cells and forms a covering called a glycocalyx that can become attached to teeth; Streptococci and lactobacilli become trapped in the dextran coating

Dental extractions or oral surgury

can lead to bacteremia and infective endocarditis (heart valves)

Synder Test Agar

Used to estimate the relative number of lactobacilli in saliva based on acid production

Yeasts

Unicellular eukaryotes, used in food and bev’s

Molds

Multicellular eukaryotes, saprophytic

Presence of crosswalls: Septate

No crosswalls: Aseptate or Nonseptate

Spores and characteristisc

Arthroconidia – rectangular, larger in size

Chlamydoconidia – round, thick-walled

Sporangiospores – formed within a sac-like structure (sporangium)

Zygomycetes (eg – Rhizopus, Mucor)

Aseptate hyphae, diploid zygospore

Basidiomycetes

Dikaryon

Dimorphism in Fungi

Two different forms of growth depending on the conditions of growth (eg – temp)

Mold form at 25 C

Yeast form at 37 C

Parfocal microscopes

A specimen in focus under the low power objectives should be nearly in focus under the oil immersion objective as well

Wavelength of light in a brightfield microscope

200 nm

Fomites

Non-living surfaces, not sterile, most bacteria grow in most temperature except super hot

Food Borne pathogens

Salmonella, Hepatitis A

Water borne pathogens

E. coli, campylobacter jejuni, vibrio cholerae, salmonella, hepatitis A

Sources of contamination

Air, Fomites, Saliva, Touch

Nutrient Agar Recipe

Peptone

Beef Extract

Distilled water

Agar

Detecting growth

Deeps: Stabbed

Slants: Streaked

Broth: Cloudiness/ turbidity

Plates: Streaked colonies, single colonies

Pure colonies

• A pure culture contains
  only a single type of
  organism.

• A pure colony arises from
  a single bacterium
  through cell division.

• Pure cultures also exist in
  broth cultures.

• TSA: nonselective.

MSA

Selective: G+(Staphylococci) only

Differentiation: Mannitol salt-
fermenting G+, (yellow if + result, no
color change if -)

Fermentation: Mannitol salt to acid.

Example: Staph aureus

MAC

Selective: G- bacteria only

Differentiation: lactose-fermenting G-
bacteria (red if + result, white if - result)

Fermentation: lactose to acids.

Example: Escherichia coli

BE

Selective: Bile-tolerant bacteria

Differentiation Hydrolysis of esculin
(black precipitate if +, no blackening of agar if - result).

Example: Priestia megaterium

India Ink

Act as a negative stain that
creates a dark, opaque
background

Examples of bacteria with capsules

• Klebsiella pneumoniae

• Haemophilus influenzae

• Neisseria meningitidis

Endospore Stain

An endospore is an oval, metabolically inactive structure that is formed within the bacterial vegetative cell. They release toxins once they become active.

Endospore Resists

Boiling water.

Chemicals

Staining.

Two genera of bacteria that produce endospores

• Bacillus (Gram-positive, aerobic
  rods)

• Clostridium (Gram-positive,
  anaerobic rods)

Endospore stain

• Stained spores appear green and the sporangium is stained pink

Malchite green and safranin

Acid Fast Stain

Mycobacterium tuberculosis

Mycobacterium leprae

Mycobacterium ulcerans

Mobility can be observed using a

1) hanging drop preparation

2) flagellar staining

3) stabbing a motility deep tube