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History of MicroBio

1. Anton van Leeuwenhoek


1st described bacteria (1677) simple microscope w/ compound
lenses
3 major bacteria forms (C,B,S)
Visualized fungi, protozoa, spermatozoa
Father of Bacteriology
2. Robert Hooke
Compound micro. 1678; confirmed 1s discoveries
3. Spontaneous Generation Theory/ Abiogenesis
Life could develop from decomposing, nonliving material
Francisco Redi (17th Cen) maggots in meat depend on
deposition of eggs by flies
Theory of Biogenesis 1st Rudolf Virchow (1858)
Life must arise from preexisting life
Also by Pasteur & Tyndall
4. Louis Pasteur
Filter microorg from air source of contamination
Fermentation of fruits & grains by ferments
Chicken cholera vaccine 1st vaccine; attenuated org.
Anthrax vaccine from herbivorous animals
Mutation at rm. temp (pathologic char. gone)
Rabies vaccine 15 passages before attenuated
5. John Tyndall
Dust carries germs
Bacterial spores killed by Tyndallization
fractional sterilization; successive heating
2 types of microorg: vegetative & spore-forming
1st day kill vegetative org. (heat at 80C, 5 mins)
2nd day spore becomes vegetative
3rd day free from microorg.
6. Roger Bacon
Franciscan monk
13th Cen Germs (invisible, living; cause diseases)
7. Germ theory of disease
By Pasteur
Specific infectious disease is caused by specific microorg.
8. Joseph Lister
Aseptic surgery spraying OR w/ aqueous phenol corrosive
change to Carbolic acid
1st pure culture solid medium key to ID of bacteria
9. Robert Koch
Perfected ID of organisms (solid media)
1876 etiological role of bacteria for anthrax (isolate in pure
culture transmit to mice)
1882 tubercule bacillus
Kochs Postulates (specific bacteria causes disease):
1. Causative agent present in disease case & not in healthy
animals
2. Isolate pathogen & grown in pure culture
3. Same disease produced microbes from pure culture are
inoculated into healthy animals
4. Same pathogen recovered from infected animal & grow again
in pure culture
Exceptions to Kochs Pos:
1. Mycobacterium leprae only satisfy no. 1
Only grows in natural, living environment (humans)

2. Virus satisfies only 1 & 3; cultured only in live cells


3. Commensals doesnt satisfy 1 pathogenic only when
resistance is low
Normal flora triggered by drugs (antibiotics)
Resistant to antibiotics
Microbio branch of Bio dealing w/ microbes; branches:
a. Bacteriology study of bacteria
Unicellular, can self-multiply & grow
Can be normal flora (skin, GIT, upper respiratory tract) or
pathogenic
Asexual spores
b. Virology study of viruses (smaller than bacteria)
Obligate intracellular parasites (use whats inside cell; cant
multiply outside cell)
Nucleic acid (RNA or DNA) enclosed by capsid (protein)
Bacteriophage capable of infecting bacteria
c. Mycology study of fungi
Yeast (single-celled) or mold (multi-celled)
Dimorphic exist as yeast & mold
Sexual & asexual
Growth cycle: vegetative & reproductive
Tinea versicolor ap-ap; ringworm (circular lesion)
Cause: Malassezia furfur (also causes dandruff)
d. Parasitology study of parasites
Flagellates
Amoeba Entamoeba histolytica
Sporozoans
Nematodes roundworm
Trematodes (flukes) flatworm
Cestodes (tapeworm) - flatworm
e. Immunology cells, molecules & mechanisms for immunity
Microbial Taxonomy comprised of 3 disciplines:
A. Classification org. that share similar morphology, physiology &
genetic traits TAXA
a. Species most basic taxonomic grp
Bacterial strains w/ common physio & genetics
Subspecies
o Biotype differ in biochemical/physio properties
o Serotype antigen-antibody
o Phagotype - bacteriophage
b. Genus comprised of different species
B. Nomenclature binomial system (Species name: Genus + specific
epithet) by Linnaeus
C. Identification microorgs key features are delineated & compared;
general categories of ID methods:
1. Genotypic char. genetic make-up (genes& nucleic acids)
a. DNA base composition ratio extent to w/c DNA of 2 orgs is
made up of cytosine & guanine relative to their base count
b. Nucleic acid base sequence analysis or homology order of
bases in DNA strand
- Nucleic Acid Hybridization complementary base pairing
2. Phenotypic char. expression of a genotype measurable in lab,
features beyond genetic level
a. Microscopic morphology size,shape,inclusion,arrangement
b. Staining characteristics
c. Environmental requirements Kochs postulates

d. Nutritional requirements utilize C & N2 sources as


nutritional substrates
e. Macroscopic morphology microbial growth patterns on
artificial media using unaided eye
f.
Subcellular properties molecular constituents typical to
taxa
g. Resistance profiles against antibiotic, heavy metal & toxin
h. Antigenic properties when it enters body & encounters
immune cells, it helps production of antibodies
Basic Cell Types
A. Protists unicellular; dont form tissues & organs; based on nucleus:
a. Eukaryotes higher protists
- Algae (red, green, brown), fungi, protozoa, slime mold
- True nucleus membrane bound
- Membrane-enclosed organelles specific functions
- Cytoskeleton, mitotic apparatus
- complex phospholipids, sphingolipids, histones, sterol
- multiple chromosomes & nucleosomes
b. Prokaryotes lower protists
- Bacteria, cyanobacteria (blue-green algae), archaebacteria (methanogens, extreme halophiles [saltloving], thermoacidophiles [acid & heat loving])
- Naked nucleus not membrane bound
- No organelles, histones
- Only Mycoplasma has phospholip, sphingolip & sterol
- Cell wall (peptidoglycan w/ muramic acid)
- Haploid w/ single chromosome
- Divide by binary fission
Microscopic morphology
I. Accdg. To ribosomal RNA (Carl Woese): 3 domains:
A. Eucarya eukaryotes, ester linked
B. Bacteria
C. Archaea CW doesnt have muramic acid, ether-linked lipids
II. Shape & arrangement
Filamentous form long strands of cells
Pleomorphic bacteria change shape
A. Coccus spherical cells
a. Diplococci in pairs
1. Streptococcus pneumoniae lobar pneumonia; lanceolate
(flat adjacent sides)
2. Neisseria gonorrhoeae coffee bean
b. Streptococci in chains
1. Streptococcus pyogenes pus producing, bacterial sore
throat, tonsillitis
c. Staphylococci in irregular / grape-like clusters
1. Staphylococcus aureus produces golden-yellow pigment,
acne food poisoning (produces toxin)
d. Tetrad in grps. Of 4
1. Gaffkya tetragena - commensal
e. Sarcina in cubical packets of 8
1. Sarcina lutea
B. Bacillus rod shaped
a. Fusiform bacillus both ends are tapered, fixed center, single
b. Diplobacilli
1. Mycobacterium tuberculosis
a) Slipping
b) Snapping
c. Streptobacilli - more than 3 cells

1. Bacillus subtilis sporulating, lab contaminant (can overgrow


causative agent, unidentifiable)
d. Coccobacilli short, thick, oval-shaped, longer than cocci
1. Escherichia coli enteric, pathogenic
e. Vibrio comma-shaped
1. Vibrio cholera
C. Spirals if you stretch very long bacillus, bacilli in helix form
a. Spirillum rigid when in motion
1. Campylobacter jejuni
b. Spirochete bends in motion; accdg. to tightness of coiling:
1. Treponema tightly coiled, corkscrew appearance
a) T. pallidum syphilis
2. Leptospira less tightly coiled w/ sharp hook-like bends at
the ends of the cells; Leptospirosis
a) L. interrogans
3. Borrelia much less tightly coiled, extremely long undulating
bacillary forms
a) B. recurrentis recurrent fever
III. Size

Micron = , micrometer = m

1 m = 1/1,000 of a mm or 1/25,000 of an inch

Cocci 0.4 2 m

Bacilli 0.2 4 m (width) x 0.5 20 m (length)


Haemophilus influenzae smallest pathogenic; meningitis(child)
Bacillus anthracis largest pathogenic

Spirals 1 14 m (length)
IV. Visualization (eye cant see <30m)
A. Microscopes
a. Brightfield / Compound Light for fungi, parasites, bacteria
- Not for viruses
- Resolution / Resolving Power detail is maintained, clarity
- Oil immersion enhance resolution prevent light rays from
dispersing after passing thru specimen
- Contrast make objects stand out from backg..; thru staining
b. Fluorescent
- FLUORESCENCE - fluors / fluorochromes (stain) energy
level absorb UV light (excitation) normal energy level
release visible (fluorescent) light; backg.: dark
a) Fluorochroming only dye; enhance contrast
b) Immunofluorescence (Fluorescent Antibody Technique/FAT)
- Dyes linked to specific antibody
- Amplified contast + antigen-antibody binding
- Ideal for spirochete staining
c. Phase contrast w/o stains
- Thickness of cell structure (refractive index) deflect light
- refractive index beam is slowed down light intensity
- Light & shadow ; for manual counting of platelets
- Advantage: viable microorg. can be observed
d. Darkfield w/o stains
- Condenser light on object only at oblique angle deflected
upward to lens dark field background
- Detect thin bacteria w/c cant be grown in culture (spirochetes)
e. Electron electron beams instead of light
- Instead of lens: Electromagnetic field form image in
fluorescent screen
- More than 100,000 magnification
a) Transmission (TEM) electron beams thru objects; for
internal structures

b) Scanning scan surface; 3D view of surface structures


B. Techniques for microscopic study of microorg.
a. Unstained, living state
1. Direct wet mount detect motile bacteria (C. jejuni, V.
cholera) & parasites
- Thru DF, PC & LM thru partially closed diaphragm
- Liquid: specimen spreads thru capillary action
- Solid suspend w/ NSS spread thru capillary action
2. Hanging drop morph. Is less distorted & motility better
appreciated than DWM
- Thru LM w/ partially closed diaphragm
- Petroleum jelly add depth/ height
3. Intravital staining dilute dye w/ no toxic effect live,
colored org.; morph. Is less distorted
b. Fixed, stained state direct specimen or growth from cultures
- Most useful for presumptive bacteria ID & virus presence &
definitive ID of parasites & fungi
1. Smear preparation
Liquid spread cells wont overlap clear morpho.
Solid w/ NSS spread
Swab roll over slide (make sure all sides touch slide)
- 1 swab inoculate to avoid contamination
- 2 swabs (1) make smear (2) inoculate
2. Air drying (free flowing air / near light)
- Preserve morpho. (kill) & allow smear to adhere to slide
3. Fixation thru:
a) Heat smear side up ensure smear affixes to slide
b) Alcohol (95% methanol) preserve RBC, WBC, bacteria
4. Staining
a) Simple stains single dye (methylene blue, crystal violet,
carbol fuchsin, safranin)
- Differentiate thru size, shape & arrangement
- Positive: org are stained (dilute carbol fuchsin)
- Negative or Relief: background is stained (India pink or
Nigrosin method)
b) Differential stains more than 1 dye
- Contrasting colors for diff. org (due to affinity)
- Gram stain & Acid Fast stain
c) Special stains color & isolate specific parts
- Metachromatc granules Albert, Loefflers Methy. Blue
- Flagellar Leifson, Grays
- Spore Wirtz-Conklin, Dorner
- Capsule Anthony, Hiss
Staining Reactions
A. Gram stain SOP (most useful & applied)
- Dr. Hans Christian Gram - 1884
a. Gram (+) retain primary stain; Deep Violet
- All cocci except Neisseria, Moraxella (Branhamella) catarrhalis
& Veillonella (anaerobic)
b. Gram (-) pink or red
- All bacilli except acid fast (Myco, Nocardia), sporeformers
(Bacillus, Clostridium) & Corynebacterium species
- Spirals (although difficult to stain)
- RBC & WBC

Steps in staining (Huckers method)


1. Make smear
2. Crystal violet primary stain; cytoplasm will be purple
3. Grams iodine mordant enhances color of primary stain,
enhance affinity to stain; CVI complex

4. 95% alcohol (absolute) or Acetone-Alcohol soln. (50-50)


decolorizer
- Gram (+) purple; Gram (-) colorless CVI washed out
5. Safranin counterstain Gram (-) pink

Principle
- Gram (+) more protein in CW alcohol coagulates protein
close pores decrease permeability trap CVI complex
- Gram (-) more lipids in CW dissolved w/ alcohol pore
size permeability CVI complex escapes
- Magnesium ribonucleate protein complex, absent in Gram (-)
- G (+) lost CW integrity due to (1) old age (2) action of
autolytic enzymes (lysozymes) (3) treatment w/ antibiotic
CV washed out during decolorization Gram variable
B. Acid fast stain sputum specimen
CW have long chain mycolic acids (50-90 C atoms)
resistance to decolorization of basic dyes by acid alcohol
a. Acid fast Myco, Noc, & Coccidian parasites (Cryptosporidum)
- Myco v. hydrophobic surface resist dyes
- Most acceptable concept: selective permeability
- To get dyes into cells:
1. Heat enhance penetration & retention of dye
2. Inc. concentration of phenol Cold Kinyoun technique
3. Inclusion of detergent Mycolic acid distorted
- Steps:
1. Make smear
2. Carbol fuchsin primary stain red
3. Acid alcohol decolorizer
4. Methylene blue - counterstain
b. Non-acid fast blue
Bacterial Cell Ultrasturctures
A. Cell envelope induce antibody response antigenic
Gram (+)
Gram (-)
Cytoplasmic membrane (inner)
Cytoplasmic membrane
Thick petidoglycan (CW)
Single planar sheet of peptidog.
Outer membrane (lipopolysach)
Variable capsule
Variable capsule (may be absent)
a. Surface adherent structures / Glycocalyx
Composition: - polysaccharide
- polysaccharide protein complex
- polypeptide
Gelatinous, viscous, poorly staining
Extracellular polymers produced by org.
Significance:
- Degree of pathogenicity (virulence) interferes w/
phagocytosis (no attachment for WBC)
- Antigenic Vi antigen & used for ID
- Glycocalyx adherence of bacteria to cell surfaces
1. Slime layer small amt.
- Surface layers loosely attached to CW diffuse into
surrounding; easily washed off
- S. lutea
2. Capsule addtl. definite surface layer, firmly attached to CW
- Visualized by negative staining halo of light surrounding
cell (India ink or special stains)
- Fresh isolates of capsulated org moist, slimy colonies

b.

c.

d.

e.

- S. pneumonia, Klebsiella pneumoniae (coccobac), H.


influenzae (coccobac), B. anthracis (mainly polypeptide
capsule), Clostridium pefringens (cause gas gangrene)
3. Glycocalyx seen as network of fibrils extending from surface
- Only by EM
- Streptococcus mutans
Outer membrane only in G (-)
Phospholipid bilayer outer phospho: lipopolysacc (LPS)
LPS consist of lipid A & a polysac (core & terminal units)
- Attached to OM by noncovalent hydrophobic bonds
- Endotoxin cause fever & shock
- Polysacc w/c extend outward outermost molecules of
OM & major surface antigen of G(-) (O / somatic antigen)
Porins scattered t/o LPS
- Control passage of nutrients & solutes
- Attachment sites (receptors) for viruses
Lipoprotein layer beneath OM; attach OM to CW; helps
anchor OM to underlying peptidoglycan
Significance:
- Carrier of O antigen & phage receptors (Porins)
- Selective permeability barrier
Cell wall / peptidoglycan / murein / mucopeptide / glycopeptides
2nd barrier against mechanical disruption
Composed of disaccharide pentapeptide subunits:
- N-acetylglucosamine (NAG) & N-acetylmuramic acid (NAM)
alternating sugar cmpds. w/ amino acid side chain linked
to NAM
- Polymers are crosslinked via peptide bridges to form
peptidoglycan sheets
- Sheets are crosslinked form Murein sacculus or sack w/c
surrounds entire cell
- Archaea CW no muramic acid
G (+) have teichoic acids (glycerol or ribitol phosphate)
surface antigens of G (+) teichoic a & CW associated protein
when dissolved by lysozyme lyse in H2O/ serum
Protoplast wall-less osmotically sensitive spherical body
stabilized in hypertonic soln. of sucrose or salts, G (+)
Spheroplast protoplast whose CE components are retained;
G (-), remnants of OM
Mycoplasma naturally wall-less, no CW, difficult to stain
Major properties:
- Protection from osmotic lysis
- Rigidity & shape to cell
- Surface antigens (G +)
- Support for propulsion by flagella
Periplasmic space only in G (-)
- Boundaries: OMs internal surface & external surface of CM
- Has gel-like substance secure nutrients from envi.
- Has enzymes detoxify solutes & degrade macromolecule
- Has membrane-derived oligosaccharides, hydrolytic
enzymes & proteins bind sugars & transport materials
Cell membrane (3rd barrier) in G (+) & G (-)
Lipid bilayer laced w/ proteins & enzymes
Osmotic barrier, functions like eukaryotic cells organelles
Functions:
- Transport of solutes into & out the cell

- Houses enzymes for OM & CW synthesis & assembly &


secretion of extracytoplasmic & extracellular substances
- Generation of chemical energy (ATP)
- Mediation of chromosomal segregation during binary fission
- Houses molecular sensors
B. Appendages
a. Flagella bacilli & spirochete, not cocci; flagellin (protein)
- Uniform length & diameter; 3 parts:
1. Filament: external to cell, connects 2 to cell surface
2. Hook: attached to basal body
3. Basal body: anchored to CM, composed of rod & 2 or
ore encircling rings (appear contiguous w/ CE)
Proteus species swarm as thin film of growth on agar
medium H antigen (flagellar antigen) from Hauch
(German) like breath condensing on a cold glass surface
Flagellar stain employs mordant w/c thickens flag.
Messeas Classification
1. Monotrichous single polar flagella
- V. cholerae, C. jejuni, Pseudomonas aeruginosa (cocco)
2. Amphitrichous both poles have flagella (2-3)
- Pseudomonas species (other than aeruginosa)
3. Lophotrichous one pole has several flagella
- Pseudomonas species
4. Peritrichous flagella all over the surface (8-12)
- Salmonella typhi, Proteus vulgaris (UTI)
5. Atrichous no filaments
- Shigella dysenteriae (cocco), K. pneumoniae
Significance:
- Rapid motility of bacteria (motility can be detected by:)
Hanging drop prep (LM)
Direct wet mount (DF or PC)
Swarming spread of bacterial growth as a film
Tubidity spreading thru semisolid agar
- Antigenically distinct ID
Phase variation switch from production of 1 antigenic
type of flagella to another
b. Axial filaments only in spirochetes
- Made up of endoflagella
- Protein fibrils wound spirally around the org, originate at both
poles & overlap at center
- Beneath OM in periplasmic space
- Flagella-like structure for motility (traveling helical wave)
- Only in EM
c. Microfibrils: Pili or Fimbriae
Hairlike structures extending from CM
Straighter, thinner & shorter than flagella
Made of pilin (protein)
Only by EM
Types:
- Common / somatic pili numerous & all over C. surface
- Sex / conjugal pili fewer, conjugation bet. 2 cells
- Both may occur independently or simultaneously
Importance:
- Common pili Adhesins (help bacteria attach to host
surfaces) or Lectins (bind specific sugars to host surface)
- Sex pili conduit for passage of DNA during conjugation
- Slow twitching/gliding motion of nonflagellated bacteria
C. Cytoplasmic structures

a. Mesosomes invagination of CM to cytoplasm


- Membrane-associated cytoplasmic sac in Gram (+)
- Has lamellar, tubular or vesicular structures
- Associated w/ division septa
- Functions:
o Form cross walls after division of genetic material
o Septal meso. DNA replication & cell division
o Lateral meso secretion; give rise to vesicles (enzymes)
b. Nucleoid Where highly coild DNA is intermixed w/ RNA ,
polyamines & proteins w/c lend structural support
- DNA = 1 mm in unfolded state
- Attached to CM or septal mesosome (often)
- Functions:
o Houses info for bacterial functions
o Direct synthesis of cellular products
o Transmission of traits
c. Ribosomes 30% protein, 70% RNA
- Site of action of antibiotics w/c inhibit protein synthesis
- For protein synthesis
d. Cytoplasmic granules/inclusion granules
Wider than cell, darker than cytoplasm
Accumulation of food reserves (storage granules); types:
- Glycogen major storage material of enteric bacteria
- Polyphosphate / metachromatic / volutin / Babes-Ernst
granules storage form of inorganic PO4
Abundant in M. tuber, C. diptheriae, Yersinia pestis (plague)
Used in ID of org.
e. Endospore distinguishing feature in aerobic Bacillus &
anaerobic Clostridium
Sporulation under adverse physical or chemical conditions &
scarcity of nutrients
Germination restoration of vegetative state; suitable envi
True endospore highly refractile body
Has Calcium dipicolinate for extreme resistance to adverse
envi conditions (radiation, heat) by making nucleic acids more
resistant to denaturation
Its core DNA & small cytoplasm w/ stable components of
protein synthesis
Classification:
- Central both ends of spore have equal amt. of cytoplasm
- Subterminal unequal amt. of stained cytoplasm
- Terminal drumstick Clostridium tetani
Importance:
- Taxonomic ID for species (constant size & location)
- Clinical resistant to sterilization & adverse envi. conditions
f. Extrachromosomal factors not essential for viability
Double-stranded DNA w/c determine bacterial traits
Plasmids extrachromosomal pcs. of DNA
In cytoplasm; autonomous or independent, attached to DNA
Transferred by conjugation plasmids have 2 genetic regions
(1-for sex pili, 2-characteristics of donor bacteria)
Types:
1. F (fertility) factor bacterial sexuality
- F+ cells w/ F factor, males, donors, sex pili
- F- cells w/o F factor, females, recipients
2. R (resistance) factor plasmids w/ genes w/c convert drugsensitive bacteria to antibiotic resistant cells

- Drug resistance: tolerance against antibiotics, by mutation


3. Colicinogenic (col) plasmids for synthesis of bactericidal
proteins (colicins)
4. Virulence plasmids genes for coding for toxin production
- Diphtheria (pseudomembrane cover air passage), tetanus
Bacterial growth inc. pop. Size (reproduction by binary fission)

Generation/doubling time time required for cell to divide


(least: 20 mins)

Bacterial growth curve:

4 basic phases of growth:


1. Lag rejuvenescence or physiologic youth
- Adaptation period
- Little or no cell division
- Not dormant intense metabolic activity (DNA & enzyme syn.)
- End: cells lose / used up their reserve storage granules
2. Logarithmic / Exponential growth (Log)
- Reproduction most active; minimum gen. time
- Cell most active metabolically
- Cells most susceptible to adverse conditions
- Majority of cells: young; gram staining right color
3. Stationary / Plateau - division = death
- Growth rate slows down, pop. size stabilizes
- Metabolic activity slows down
- If not transferred to new medium metabolic waste
accumulates food stuff depleted, change pH (acid kill bac)
4. Death / Logarithmic decline death > growth
Nutritional requirements
1. Carbon
a. Autotroph (lithotroph) CO2 as sole source of C C skeleton
- Only H2O, inorganic salts & CO2 for growth
b. Heterotroph (organotroph) require C (not from CO2) in
organic form
- Organic molecules (glucose) as electron donors
- Pathogenic bacteria
2. Nitrogen in either organic form
- Synthesis of nucleic acids (DNA, RNA) & proteins
3. Inorganic ions
a. Sulfur synthesis of S-containing AA & vit. (thiamine, biotin)
b. Phosphorus synthesis of NA & phospholipids of CM
c. Potassium, magnesium, calcium cofactors for enzymes
- Mg stabilize ribosomes, CM & NA
d. Iron, copper, molybdenum, zinc trace elements as cofactors
4. Growth factors cant be synthesized
- In culture as yeast extract, whole blood or serum
a. B complex vit. coenzyme
b. Amino acids protein manufacture
c. Purines & Pyrimidines NA synthesis
Environmental requirements
1. Oxygen
a. Strict aerobes require O2 to grow
- Breathe toxic mat. forms 1st rad: superoxide superoxide dismutase 2nd rad: H2O2 catalase H2O & O2
b. Strict anaerobes not grow in presence of O2 (can be killed)
- No superoxide dismutase; C. tetani
c. Microaerophiles only in low levels of O2
- Limited tolerance: sensitivity to superoxide free radicals

d. Facultative anaerobes / aerobes can frown anaerobically


ferment carbs lactic acid & acetic acid (fermentative met.)
- w/ air oxidative metabolism carbs H2O & CO2
- pathogenic bacteria
e. Aerotolerant anaerobes fermentative met., no oxidative met.
- Have superoxide dismutase neutralizes toxic O2
2. Carbon dioxide (5-10%) initiates growth
- Capnophiles Neisseria gonorrheae & meningitidis
3. Moisture 75-80%; H2O major component of cytoplasm
- Dissolves food materials in envi.
- Major constituent of culture media
4. Temperature minimum, optimum & maximum
5. pH clinically relevant: near neutral (6.5-7.5)
- acid or alkaline pH for preserving food (pickles, cheese)
- V. cholerae 9.6; Fungi 5.0
6. Ionic strength & osmotic pressure killed/inhibited (H2O goes out
of bacteria) by conc. Of salt or sugar
- Gram (+) osmotically tolerant
- Basis in preserving food
- Vibrio parahaemolyticus 3-7% NaCl
Temp type:
Found in
Range
OT
Psychrophilic/
Cold H2O & soil; cold-loving
-5 - 30
10 - 20
cryophilic
Spoilage of food in fridge
Middle temp; pathogenic (37)
10-45
20-40
Mesophilic
Hot springs, rotting compost
25-80
50-60
Thermophilic
piles, tropical soil, hot H2O
heaters, hot tubs, thermal vents
Laboratory Cultivation & Isolation of bacteria

Growth for definitive ID

3 main purposes:
1. Grow & isolate all bacteria in an infection
2. Determine w/c bacteria is most likely causing the infection
3. Sufficient growth of clinically relevant bacteria for ID

Principle
Cultivation taking bac from infection by specimen collection
& growing them in artificial environment
Culture org. that grow & multiply in / on a culture medium
Classification of culture media (physical state, composition,
use)
(******************)
Preparation:
Dehydrated (powder / granule) or tablet
Reconstitute w/ H2O
Agar containing media heated to dissolve agar
- Boiling H2O bath instead of direct heat
Containers:
- Petri dish (plate) solid medium; large surface area
- Test tubes solid, semisolid, liquid media; covered w/
cotton plug (easy exchange of air) or screw cap (loosened)
Butt preparation:
Butt
Slant
Slant-butt

Petri dish media sterilized before dispensing into plate


Test tubes sterilization after dispensing
Sterilization
Should be sterile before use (no signs of growth)

1.

2.
3.

Most common: autoclave (121C) some cant w/stand, so:


Inspissation media w/ serum or proteins (Loefflers agar)
Filtration carb. soln. w/c may be denatured by heat
Quality control
Check for sterility
Test w/ QC org. of known physiologic & biochem. Properties
If theres growth after incubation discard batch of media
Storage
Common: refrigeration prevent dehydration & deterioration
- Fridged media must come to room temp. before using
- Dehydrated separates from tube
Process of bacterial cultivation
Inoculation implantation of specimen (inoculum) into media
- Sterile cotton swab, inoculating wire loop/needle
- Inoculum to be introduced to:
a. Liquid media (broth) suspend & mix
b. Solid media:
Butt stab
Slant line streak
Slant-butt line stab streak
Incubation proper temp & ventilation
- 18-24 hrs. at 37
Inspection of cultures (w/in hours)
a. Indications of growth in a broth
Turbidity ask for uninoculated & compare
Change in color produce pigment
pH change
Gas bubbles: aerogenic (gas producing) bubbles on top
b. Indications of growth on solid media form colony char:
a) Size (use ruler / vernier caliper) in mm
- Pinpoint, small, medium or large
b) Pigmentation color w/ no pH indicator
c) Shape:
Form punctiform
- Circular
- Filamentous (cottony)
- Irregular
Elevation flat
- Raised
- Convex (dome shaped)
Margin entire
- irregular
d) Surface appearance
Glistening - shiny
Dull
Translucent light passes thru; w/o color
Opaque w/ color
e) Consistency & texture
Dry & friable can be pushed around
Viscous clings to wire
Smooth homogenous, glistening
Mucoid waterlike, confluent appearance (capsule or s.
layer); if kept in long time smooth longer rough
Rough striated or granular
f) Changes in agar media
Hemolytic pattern on blood agar clearing of area around
colony; lysing RBC

Color change in pH indicator


Pitting
g) Odor - just open a little bit
Pseudomonas aeruginosa grape like smell

Method of obtaining pure (axenic) culture only 1 org; solid


media
a. Streak plate most practical & most useful
- Mixed pop (w/ normal flora from GIT) pure culture upon
subculture (Single Colony Sub-Culture / SCSC)
a) Simple streaking
b) Multiple interrupted streaking respiratory tract
c) Four quadrant streaking more isolated colony

b. Pour plate (colony ct) or spread plate (solid media)


- Determine approximate no. of viable org. in liquid (H2O & milk)
- Result: no. of colony forming units (CFU) per ml (only live bac
form colony)
- Not a good way of obtaining culture
- Aliquot in sterile container mix allow to solidify
Other methods for Lab Diagnosis of Infectious Disease
A. Biochemical test (conventional)
a. Enzyme based test (catalase, urease, oxidase, PYR, indole)
b. Presence of metabol. pathway (deamination, decarboxylation)
c. Inhibitor profiles
B. Molecular methods for ID & characterization expensive
a. Nucleic acid based hybridization
- Target nucleic acid amplification
o Polymerase Chain Reaction (PCR)
b. Non-nucleic acid based analytic methods
- Chromatography (GLC)
- Electrophoretic protein analysis
C. Immunochemical methods for org. detection antigen-anti-B rxn
- Known antibody; detect what antigen is present
- RIA, ELISA, precipitin, EIA
D. Serologic diagnosis of infectious disease known antigen
- for presence of antibody
- agglutination, ELISA, FAT, RIA, Western-blot, ppt. test
E. Detection of antibacterial resistance antibiotic susceptibility
a. Broth dilution (conventional); broth microdilution (commercial)
1. MIC (minimum inhibitory conc) least amt. of antibiotic that
can inhibit
2. MBC (minimum bactericidal conc) least amt. of antibiotic
that can kill avoid overdose
b. Agar dilution
c. Disk diffusion
F. Use experimental lab animals mice, guinea pigs, monkey, pigs
G. Epidemiology
a. Bacteriophage typing needs many mat.
b. Serological typing only needs sample, slide & rgt
- Identify strains (serotypes)
- Mix drop of antiserum w/ specific antibody clumping
Variation
Deviation form parent form (under diff. conditions)
Temporary / permanent
A. Variants may appear as a result of:
a. Spontaneous mutation change in genetic make-up w/in org

b. Induction by environmental factors exposure to toxic,


radiation, prolonged growth on artificial medium
B. Types
a. Morphologic
1. Pleomorphism many forms (size, shape) (Corynebac.)
- Represents dying / transfer to another medium
2. Dissociation change colony type formed on semisolid m.
3. Cell structures
a) Capsule diminish, restored by animal passage
b) Flagella no. & location remain constant
c) Spores affected by envi.
d) L-forms w/ defective / absent CW transparent
b. Physiologic
1. Adaptation modification of org. to make it fit for existence
Attenuation loss in disease producing ability
- In highly pathogenic temp / permanent
- vaccines (BCG Bacillus of Calmette & Guerin)
- vaccine of M. Tuberculosis use live attenuated
adv: antibodies are retained
disadv: org. can return to pathogenic state
c. Genetic
1. Mutation change in genotype & phenotype
- Base substitution / Pt. mutation 1 DNA base change
- Frameshift mutation deletion/insertion of nucleotide pairs
- Can be spontaneous (w/in org) during division
- Can be induced by chem/physical factors
- Product: mutant
2. Gene exchange/transfer & recombination
a) Transformation (primitive)
- Recipient c uptake DNA of dead (lysed) bacterial c
- Competent c use DNA to transform
- Haemophilus, Streptococcus, Neisseria, Bacillus
- Devt. of antibiotic resistance & dissemination of genes
(encode for org.s ability to causes disease)
b) Transduction involves virus
- Mediated by bacteriophage
- Genes enclosed in capsid during virus replication
- Transducing particles viral capsids w/ bac. genes
1) Generalized whats injected is viral DNA
- Transducing p infect susceptible cell destruction
2) Specialized fragment of DNA w/ viral DNA
- Virus host chromo excision pus (viral DNA)
c) Conjugation donor has F factor (plasmid); outcomes:
1) F+ donors free in cytoplasm integrated F factor
F- F+; only plasmid is transferred
2) Hfr donors (high frequency of recombination)
integrated into bacterial chromo
- Chromosomal genes are transferred
- F (hybrid) adjacent bacterial genes + free F factor
Sterilization & Disinfection

Sterilization - all forms of microbial life (spores) are killed


- Physical: incineration, dry heat, moist heat, radiation
- Chem: Eth. Oxide, formaldehyde, glutaraldehyde

Disinfection pathogenic org (not all) are destroyed


- Physical: boiling, UV light (non-ionizing radiation)
- Chem.: alcohol, aldehyde, halogens, metals, phenolic

Biocides (chemical sterilants) (Cide/Cidal killing action


- Chemicals that destroy all life

Disinfectant chem.. sterilants for short time period

- X org w/ waxy coats


Antiseptic disinfectant on living tissue (skin)
- Lower toxicity than disinfectants

Preservatives prevent food, serum & vaccine deterioration

Sanitation lowers bacterial content of utensils used for food

Fumigation - liberation of fumes or gases to destroy insects

Deodorants mask offensive odors; obscure, not destroy


infectious

Asepsis prevent microorg. from reaching protected envi.


Factors influencing activity of disinfectants:
1. Types of orgs. present
2. Temp & pH of process (10- x2 rate of disinfection); non-ionized
pass thru CM readily than inactive
3. No. of orgs. present / microbial load
4. Conc. Of disinfectant
5. Amt. of organic material present (serum, blood, pus make
disinfectant inert)
6. Length of contact time
7. Type of H2O available (hard reduces killing rate of disinfectant)
Phenol coefficient no. on all new disinfectants
Phenol standard reference material
Highest dilution of disinfectant that kills org in 10 mins
Highest dilution of phenol w/ same result
>1 = stronger than phenol; <1 = not as strong as phenol
Antimicrobial physical agents
1. Heat cheap, generally used

Thermal death pt. lowest temp at w/c all microorg in liquid


suspension will be killed in 10 mins.

Thermal death time minimal length of time in w/c bacteria in


liquid culture will be killed at given temp

Decimal reduction time (DRT or D value) time (mins) in w/c


90% of pop of bac at given temp will be killed
a. Moist heat kill by coagulation of proteins (irreversible) by
breakage of H bonds; faster w/ H2O
1) Steam under pressure autoclave
- 121 (15 lbs steam pressure/in.sq) or 126 (20 lbs s.p/in.sq)
for 15-20 mins sterilize media liquids & instruments
- Flash autoclave saturated steam at 134 for 3 mins (OR)
- 132 (30-60 mins) infectious medical waste
- QC: B. stearothermophilus in vials / Spore-strip-set test
2) Boiling 15-30 mins at 100 - kill vegetative forms
- + 2% NaCO3 or detergent - killing effect
3) Tyndallization fractional sterilization
- 30 mins. at 80 or 100 for 3 consecutive days
- For heat sensitive culture media (w/ carbs, egg, serum)
4) Pasteurization destroy disease-producing org
- Render milk safe
- 62.9 (63) for 30 mins classic pasteurization
- 71.6 (72) for 15 sec High temp short time (HTST) past.
- 135 for 1 sec or 151 <1 sec Ultrahigh temp (UHT)
- 70 for 30 mins inhalation therapy equipment free of org
- Rapid cooling if not: destroy nutritional value
b. Dry heat no H2O; temp, longer protein oxidation
Lethal: level of electrolytes
1) Hot air sterilization ovens; carbonization (kill spores also)
- 160 180 for 1.5-3 hrs
- Sterilization of glasswares, oil, petroleum, powder
2) Incineration intense form for infectious waste

- 870 - 980; for emergency sterilization, needles, coverslip


2. Radiation
a. Ionizing H2O ionization OH radicals + DNA kill cell
- Cathode, gamma & x-ray pharmaceuticals & disposables
- Low gamma or x-ray dose preserve fresh fruit, veg & pork
- Short WL & high energy gamma rays - microwaves
b. Non-ionizing UV light, cheap; thymine dimmers block
passage of replicating enzymes; irradiation of air
3. Filtration not killing, but removing bac from fluids
- For antibiotic solns, toxic chem., radioisotopes, vaccines &
carb
- Liquid: cellulose acetate or cellulose nitrate w/ vacuum
- Air: high efficiency articulate air (HEPA) filter remove org
>.3m from OR
4. Freezing not for sterilization protein denaturation & CM damage
- Preservation of bacterial culture
- Deep freezing - -50 - -95
- Lyophilization - -54 - -72 dehydrated in high vacuum
5. Ultrasonic vibrations protein denaturation & coagulation
- Noiseless; Treat sewage H2O disrupt virus, bac & chem
6. Lasers medical instruments, clear OR air
Antimicrobial chem. agents
A. Agents that damage CM
1. Surface active disinfectants reduce surface tension
a.
Soaps & detergents mechanical removal of microbes
(scrub)
- More effective against Gram (+) denature protein
b.
Benzalkonium chloride (zephiran chloride) quaternary
ammonium compound, more effective on Gram (+)
- Reduce bacterial counts in food processing industries
- Disinfection of hands & prep field of operation
- 1:1000 dilution (more effective) kills vegetative in 30 mins
(except TB)
2. Phenolic cmpds
a.
Phenol carbolic acid
- conc disrupt CM & ppt. proteins
- 0.1 2% - distort CM leakage of metabolites
- Inactivate bacterial spores
- Disinfecting feces, blood, sputum
b.
Cresol commercial: Lysol (cresol + soap)
- Objects contaminated w/ TB
c.
Substituted phenol
1) Hexylresorcinol & hexachlorophene (cause brain damage in
kids removed) less toxic; in detergent - germicidal
action of surface-active agents (hexachlorophene + soap =
pHisoHex, Hexosan)
2) Chlorohexidine replace hchlorop., non-toxic, not phenolic,
hand scrubbing & preop skin prep
d.
Alcohol ethyl alc., isopropyl (more germicidal, toxic)
- Benzyl alc preservative
- Germicidal action = MW; MW = GA
- Tuberculoidal kill M. tuber.
- Most effective: 50 - 70%
B. Agents that denature protein unfolding of polypeptide chain
1. Acids & alkalies change pH
- Eyewash (boric acid), anti-fungal (salicylic acid),
preservatives (benzoic, lactic, propionic, acetic, citric)
2. Alcohols germ. Action protein coagulation

C. Agents that modify functional grps of protein & NA (on enzymes,


CW, CM)
1. Heavy metals enzyme inactivation
a. 1% AgNO3 against opthalmia neonatorum (infants), warts
b. Mercuric chloride ppts. Sulfhydryl by enzyme; wood, paper
c. Merthiolate (thimerosal) preserve vaccines; skin antiseptic
d. Merbromin (mercurochrome) & nitromersal (metaphen) skin
antiseptic
2. Oxidizing agents
a. Halogens
1) Chlorine sanitize drinking H2O
- Irreversible oxidation of sulfhydryl on enzymes
inactivate enzyme (changed tertiary struct.)
2) Iodine - reacts w/ tyrosine prevents fun. enzymes w/ Tyr
- Skin antiseptic
b. Hydroxide peroxide causes tissue damage; mildly antiseptic
3. Dyes combine w/ proteins, interfere w/ repro mech. of cells
- Mumps, against Gram (+)
4. Alkylating agents
a. Aldehydes
1) Formaldehyde attaches to proteins & NA - nonfunctional
a) Gas disinfectant, bactericide, fungicide
b) Formalin 37%, aqueous; tissue preservative
- athletes foot; things w/c cant be autoclaved
2) glutaraldehyde Cidex
- 10x more effective as bactericide & sprocide
b. Ethylene oxide laughing gas
- Killing agent for bacteria, spores, molds, viruses
- Impt. property: can penetrate into any substance
- Disadv: highly flammable
- Carboxide (10% EtO, 90%CO2), Oxyfume (20,80)
- React w/ free carboxylic, amino, SH & OH grps replaces
labile H w/ hydroxyethyl radical
Action of chemotherapeutic agents

Chemotherapeutic agents when taken orally or injected


destroys microorg w/o injury to body cells

Antibiotics produced by microorg inhibit growth of bacteria


& kill bacteria & other microorg; produced by chemical synthesis

Spectrum range of antimicrobial activity


a. Broad many microorg. affected (Gram + & -)
- Chloramphenicol, ampicillin, tetracycline, rifampicin
b. Narrow few orgs affected
- Penicillin, streptomycin, erythromycin, vancomycin
Desirable prop of antibiotic (should be/have):
1. Selective toxicity only infective bac.
2. Bactericidal - killing rather than inhibitory
3. Susceptible org dont become genotypically or phenotypically
resistant
4. Not allergenic, large doses dont cause adverse side effects
5. Active in presence of plasma, body fluids or exudates
6. H2O-soluble & stable, bactericidal levels should be rapidly reached
& maintained for prolonged periods
Mode of Action (book)
Side effects:
1. Hypersensitivity or allergy (most common)
2. Toxicity
a. Aplastic anemia chloramphenicol
b. Deafness & vertigo streptomycin
c. Dental defects tetracycline in young children & infants

d. Toxicity to liver INH, rifampin


3. Effects of replacement flora Superinfection
Oral antimicrobial drug suppress normal GIT flora
overgrowth of staphylococcus enterocolitis
Suppress normal vaginal flora overgrowth of fungus
(Candida albicans) vaginitis
4. Induction of bacterial resistance frequent antibiotic use; cause:
a. Plasmid mediated R factor
b. Mutation
1) Alteration of drug binding sites no entrance
2) Alteration of membrane permeability
c. Produce enzyme w/c destroys or inactivates antimicrobial agent
(Penicillin Beta lactam ring destroyed by Beta lactamase)
d. Produce multidrug resistant pumps pump drugs out of cells
before it damages cells