N Microbiology Notes

- Most bacteria are stained

o Exceptions include Legionella, Mycoplasma, Chalmydia and others

Gram stain mechanism

- Difference in the microbial cell wall are visualized
o Cell wall of gram negative have higher lipid content than gram positive cells

- Crystal violet penetrates both types

- Iodine is added, form the crystal violet iodine complex
- Decolorizer dissolves the lipid layer from the gram negative cells allowing CV-I complex
out

Gram stain
- Only partial bacterial identification
- Some organisms do not stain
- No organisms seen does not rule out infection

Cocci
Positive gram stain
- S. aureus, coag neg Staph, group A and B strep

Negative
- Neisseria

>25 epithelial cells/lpd = saliva

Bacteria are prokaryotes – single cell organisms lacking membrane bound nuclei

Eukaryotes are organisms with a defiend nucleus

Fermentation
- Glucose is converted into pyruvate
o Embdem Meyerhof pathway
 - Pyruvate enters Kreb’s cycle
- NADH and FADH enters the

Bacterial cell wall

- Main constituent is complex polymer – peptidoglycan
o Many sugar molecules linked by small peptide chains
o Peptidoglycan is only found in bacteria

Gram positive cell walls have a thick layer of peptidoglycan

Gram negative walls have much thinner layer of peptidoglycan, and an outer membrane

Within the cell wall of gram negative bacteria is lipopolysaccharide

Glycocalyx is a slimy, gelatinous material produced near the cell membrane and secreted
outside of the cell wall

Two types of glycocalyx

- Slime layer
- Capsule

Slime layer – not highly organized, not firmly attached

- Pseudomonas and Staphylococcus

Capsule – highly orgniazed and firmly attached to the cell wall

Flagella
- Enable bacteria to move in a liquid environment
- Consists of > three protein appendages twisted together

Pili
- Usually on external surface of gram negative
- Much thinner than flagella, rigid structure, no associated with motility
- Cytoplasm and extend through the plasma membrane, cell wall and capsule

Spores
- Some bacteria form thick walled structures
o Bacillus and Clostridium
Virulence factors
- Bacteria that cause disease are termed pathogenic due to various factors
o Exotosins
o Endotoxin
o Capsules

Myobacteria
- Group of organisms with cell wall similar to gram positive bacteria
o Cell wall contains mycolic acids
o Staining characteristic – acid fast

Rickettsia and Chalmydia

- Gram negative type cell wall
- Not free living
o Survive and multiply with host cells as intracellular pathogens

Chlamydia most primitive bacteria

- Do not have enzymes required to perform many essential metabolic activities
- Obligate intracellular pathogens
- Two species of chalmydia
o Chlamydia trachomatis and C psittaci

Myoplasma
- Smallest of cellular microbes

Types of media
- Bacteriology
o Routine
o Fastidious
o Anerobes
o Mycoplasma/Ureaplasma
- Mycobacteriology
- Mycology
- Virology
o Viruses
o Chlamydia

Constituents of media
- Agar
o Gelatinous seaweed extract
 o 1-2% agar in plates
o Nutrients
 Hydrolyzed proteins
 Animal
 Plant

Enrichments
- Yeast extracts, blood

Buffers
- Stable pH for growth

pH indicators
- Neutral red – red to colorless
- Phenol red – yellow to red
- Thymol blue – yellow to green/blue

Constituents of media
- Inhibitors
o Dyes
 Crystal violet, eosin and methylene blue
o Bile salts
 Sodium desoxycholate
o Antibiotics

Enrichment media
- Encourages growth of a particular orgniams by prodiving specific nutrients needed by
that oarganism

Supportive
- Allows most non fastidious organisms to grow at a natural rate

Selective
- Contains inhibitory agents to all organisms except one being sought
- Select for certain organisms to the disadvantage of others

Differential
- Allows organism to be morphologically distinguished from other oganisms wit hdifferent
characteristics.
Supportive
- Blood agar
- Chocolate agar
Selective/differential for gram negative bacilli
- MacConkey
- XLD or Hektoen for stool

Blood agar
- Casein peptones
o Group of proteins from milk
- Soybean peptones
- 5% sheep blood

MacConkey
- Peptone base with lactose, crystal violet and bile salts

MacConkey
- Lactose positive
o E coli
- Lactose negative
o Proteus – swarming

Xylose lysine desoxycholate (XLD)

- Yeast extract
- Sodium deoxycholate
- Inhibits gram positives

XLD
- Yellow – fermet the excess carbohydrates, cause large pH drop, yellow
- Colorless or red – no fermentation, no H2S
- Red w/black center
o Fermetns xylose, produce low pH, then decarboxylate lysine, produce high pH

Hektoen Enteric (HE)

- Meat peptones and yeast extract

- Bile salts – inhibit gram positive organisms
 - Lactose, sucrose, salacin
- Indicators – bromophenol blue and acid fuchsin
- Ferric ammonium citrate for h2S production

- Differential for salmonella nad shigella

Yellow – organe colonies = lactose fermenter (E coli

Colorless/green colonies with unchanged medium = nonlactose fermenter

Black colonies = H2S production (salmonella)

Selective/differential for gram positive

- CAN
- PEA

Colistin nalidixic acid

- Casein peptones
- Digest of animal tissue

Phenylethyl alcohol agar

- Casein peptones
- Soybean peptones
- 5% sheep blood

Key media for routine anaerobic

- Supportive
o Blood agar
- Selective for anaerobes
o Supplemented phenylethyl alcohol blood agar

Kanamycin vancomycin laked blood agar

- Aminoglycoside helps separate aerobes and anerobes in mixed cultures
- Limit the swarming of proteus spp.
- Kanamycin and vancomycin permitting growth of only gram negative anaerobes

Back up broth
- Broth medium serves as a check for agar plates
- Useful when the primary are no growth
 - Chopped meat glucose or thioglycolate broth are most common
3-5% CO2
Room air

Family staphylococcaceae

Gram positive cocci in clusters or tetrads

- Catalase positive
- Aerobic to facultative anaerobic
- Nonmotile

Genus staphylococcus
- Differentiated by coagulase test

- Humans are natural reservoir for S. aureus

o Asymptomatic colonization is far more common than infection

- Colonization of nasopharynx and perineum skin occurs shortly after birth and recurs

Epidemiology
- Carriage rates from 25% to 50%
o Higher in injection drug users, diabetes, dermatologic conditions, long term
indwelling intravascular catheters
- Young children have higher rates
- Colonization may be transient or persistent.

Toxic shock syndrome

- TSST-1 toxin associated

o Superantigen
- Characteristic rash
- Multisystem disease – high fever, hypotension and shock
- Identified in both sexes
o Higher prevalence with tampon use
- Most patients recover 2-5% mortality

S. aureus
- Carriage of the organism  Disseminated via hand to body sites and breaks in the skin
- Eczema or minor dermatitis
- Abscess  fibrin wall around a core of organisms and leukocytes
Heat stable exotoxins that cause diarrhea and vomiting

Entertoxins A and D are resistant to gastric and digestive acids

- Toxins are preformed in foods
- Symptoms appear 2-8 hours after ingestion and resolve within 8 hours

Hemolysins
- Alpha hemolysin – destroys RBCs, platelets, tissue
- Beta hemolysin – destroys RBCs
- Delta hemolysin – causes injury, less letal

Coagulase – causes coagulation of surroundings

Hyaluroniase – hydrolyzes hyaluronic acid in connective tissue
Lipase – aids colonization by acting on sebaceous glands
Fame – breaks down anti staphylococcal lipids made by the host.

Normal flora of the mucus membranes of the urogenital tract

Staphylococcus saprophyticus
- Causes urinary tract infection
o Young sexually active women
o Considered significant in urine cultures even if it is found in small number

Spore forming
- Bacillus species

Non spore forming

- Listeria, Erysipelothrix, Corynebacterium and others

Bacillus anthracis
- Most virulen and significant human disease

Bacillus cereus
- Most common disease is food poisoning
- Less common opportunistic infection
Bacillus sp. : spore formation
- Endospores are unique to Bacillus sp. Among aerobes
- Transition from vegetative cells to spores under harsh and desiccated environments
preserves cell viability for long periods of time.

Bacillus anthracis
- Zoonotic disease in herivores follow ingestion of spores in soil
- Less typical is infection nthrough intetnioanl exposure

Clinical presentation “anthrax”

- Cutaneous: direct contact with infected material
- Inhalation: aspiration of spore aerosol

Bacillus cereus:
- Common agent in food poisoning

Listeria

- Appear on gram stain as

o Gram positive short bacilli or coccobacilli
- Grows aerobically
- No spores
- Not acid fast

Six species, type species is L monocytogenes, the most important to human disease
Found widespread in nature, habitat is soil and decaying vegetation, but carried by numerous
humans and animals

Most virulent of the listeria and common human pathogen is listeria monocyotgenes

Diagnosis: Literia monocytogenes

- Gram stained smear of normally sterile body fluids with characteristic morphology
- Culture recovery from appropriate specimens

Recognition of aerobic gram positive bacilli

- Beta hemolytic
- Biochemical profile
- Motility characteristics
Enterococcous
- Gram positive cocci typically in pairs and short chains
- Normal flora of the gastrointestinal and urogenital tract of humans and naimals
- UTI

Virulence factors
- Fimbriae – attachment to epithelial cells
- Adhesins- attachment to intestinal tract
- Bacteriocins – hydrolizes collagen, hemolgobin

Laboratory diagnosis

- Small, white colonies

- Alpha or non hemolytic maybe beta
- Both enterococcus and group D are bile esculin positive

- Resistant to cephalosporins

- Small gram positive bacilli

- Occur singly, in short chains

Corynebacterium
- Small gram positive bacilli
o May resembling “Chinese letters”
- Significant human pathogen is C. diphtheriae

Invasion: bacteria colonize and proliferate in local tissues of the throat creating
pseudomembrane

Toxigenesis:
- Bacteria produce an exotoxin which causes death eukaryotic cells and tissues

All mycobacterial will stain “acid-fast” with Ziehlneelson or auromine/rhodamine stains and are
commonly referred to as AFB.

Culture media
- Slanted media in tubes preferred for safety reasons
 o Egg based (whole or yolks)
Enzyme catalase breaks down H2O2 into oxygen and water

Method
- Small loopful of organism onto slide
- Add 1 drop 30% hydrogen peroxide
- Observe for bubbles
- Positive control – S. aureus
- Negative control – Streptococcus species.

Listeria
- Tumling motility, umbrella

Erysipelthorix
- Non motile
- Test tube brush growth in gelatin H2S positive in TSI

B anthracis

- Bamboo shoots

Bacillus
- Ground glass colonies

Gardnerella vaginalis
- Clue cells

Gram negative rods

- Enterobacteriae
o Reduce NO3 to NO2
o Most oxidase negative

- Escherria Coli
o Indole positive and lactose positive.
o 0157 like Shigella

Shigella
- Lactose negative
- Indole negative
Kleb
- Opportunist

Enterobacter

- Ornithine positive

Serratia
- May produce red pigment

Salmonella
- Large number needed for infection
- H2S positive
- Lysine positive

Citrobacter
- Similar to Salmonella

Proteus
- Urea positive
- Deaminase positive
P mirabilis
- Most sensitive to penicillins
- Indole negative

P vulgaris
- Indole positive

Plesiomonas
- Oxiase positive
- Reclassified into Enterobacteriaceae

A/A – SEEK – Serratia, E Coli, Enterobacter, Klebieslla

Brucella – gram negative coccobaciilus – Brucellosis

Brucella abortus (cows) eat fuschin

Brucella suis (pigs) eat thionin
Brucella melentensis (goats) eat fuschin and tionin

Fraciesella Tulearensis – Gram negative coccobacillus

Cysteine glucose media
H2S with acetate

Yersinia the pin

- Bipolar staining

Pasteurella Multocida
- Bi polar staining
- Oxidase and Indole positive
- Ferments glucose and surcrose
- Urease negative
- Mousy odor

Streptobacillus moniliformis
- Pleomorphic
-