By: Ernest Archival B. Santos, RMT

A. Morphology1. Bacilli-rods2. Cocci- spheres3. “Spiral” shape

a. vibrio- straight rod woth single rigid curveb. spirillum- rigid helical rodc. spirochete- flexuous helical rod

4. Pleomorphic organisms vary in size and shape

B. Arrangement1. Pairs- diplococci, diplobacilli2. Chains- streptococci, streptobacilli3. Grape-like clusters- staphylococci4. Groups of four- tetrads (Peptococcus)5. Packets of eight- cuboidal (e.g. Sarcina)6. Palisades (e.g., Corynebacterium)

C. Size1. Unit of measurement- micrometer2. Haemophilus- smallest pathogenic bacillus (0.2x.05um)3. Bacillus anthracis- largest pathogenic bacillus(1x 3-10 um)

D. Capsule: mucilagious envelope that surrounds some bacteria1. May increase virulence by preventing phagocytosis2. Antigenic (basis of serotyping by quellung reactions specific capsular swelling with type-specific antiserum)3. Colonies often slimy (mucoid)4. Some organisms with capsulesa. Streptococcus pneumoniae, Klebsiella pneumoniae, Haemophilus influenzae, Neisseria maningitidis.

E. Metachromatic (dark-staining) Granules1. Affinity for basic dyes2. Special stains- Albert, Neisser, Ponder, methylene blue3. Example: Babes-Ernst granules of Corynebacterium diptheriae

F. Motility1. Motile bacteria (except spirochetes) possess flegellaa. atrichous- no flegellab. monotrichous-single flagellum at one polec. amphitrichous-single flagellum at each poled. lophotrichous-tuft of flagella at one or both polese. peritrichous- flagella all over organism2. with some exceptions, motility differentiates Shigella (-) from Salmonella (+)

3. Demonstrate motility at 18 hours growth (a young culture)

4. Often motility can be seen best at 25C (35-37C may be inbitory)

G. Reproduction1. Asexual reproduction by transverse binary fission2. Growth cycle

a. lag phase- increase in cell size Not in #b. log phase-increase in growth rate(cell

division) - susceptible to antimicrobial

agents

c. stationary/plateau phase-no net growth

d. death phase/period decline- increase death rate

H. Oxygen requirements1. Aerobe- grows in the presence of atmospheric (free) oxygen

a. obligate aerobe-cannot grow in absence of free oxygen

b. facultative aerobe- fundamentally anaerobe but can grow in presence of atmospheric oxygen

c.microaerophile- requires reduced amount of free oxygen

2. Anaerobe- grows in the absence of atmospheric (free) oxygen- containing compounds.

a. obligate anaerobe-cannot grow in the presence of atmospheric (free) oxygen

b. facultative anaerobe- fundamentally an aerobe but can grow in the absence of atmospheric (free) oxygen.

3. Capnophilic- growth enhanced by increased CO2.

J. Thermal requirements1. Psychrophilic- (“cold-loving”)-grow below 10C2. Mesophilic (“most pathogens”)- grow at 20-40C3. Thermophilic- (“heat-loving”)-grow at 50-55C

K. Hemolysis1. Alpha- incomplete hemolysis2. Beta-complete hemolysis3. Gamma- no hemolysis

L. Sterilization- complete destruction or removal of living forms 1. Moist heat- coagulase protein

a. boiling (100C for 15-30 minutes) kills all vegetative organisms but not all spores or viruses.

b. fractional- alternate heating (kills vegetative cells), incubation (spores germinate)heating (kills remaining vegetative cells)

1. tyndalization- flowing steam for 30 minutes on 3 successive days

2. inspissation- 75-80C for 2 hours on 3 successive days

3. pasteurization- used to sterilize milk or serum

c. autoclaving: steam (121C at 15 lbs psi for 15-30 minutes)

1. most effective method of sterilization2. kills all organisms3. used whenever possible to steriliza

liquid media, instruments, glasswares.4. Q.C.- Bacillus stearothermophilus

incubated at 56C.

2. Dry heat- kills by oxidationa. hot air oven- 160-180C for 1-2 hours.Q.C. Bacillus subtilis incubated at 35-37C.b. inceneration- waste disposalc. cremation- control diseased. flaming- to sterilize inoculating loop

3. Filtrationa. asbestosb. membrane filter-HEPA filter

4. Ethylene oxide (cold sterilization)a. used for materials that cannot be

autoclaved (ex. Rubber goods, plastic tubes)

b. strict requirements of concentration, humidity

c. Q.C.- Bacillus subtilis incubated at 35-37C

5. Disinfectants and antisepticsa. disinfectatnt-microbicidal

1. phenol coefficient2. quarternary ammoniums (“quats”)3. bleach

b. antiseptic-microbiostatic- “remains the same”

1. alcohol- 70% is effective than 95%2. tincture- alcoholic iodine3. iodophor-iodine detergent

M. Staining1. Dyes- many coal tar derivatives

a. basic dyes- greater affinity for nucleic. acid dyes- greater affinity for

cytoplasm2. Mordant-increase affinity or binding of dyes3. Smear “fixed” in flame4. Simple- one stain5.Differential stains- more than one stain

Purpose Reagents Gram (-)

Primary V (crystal violet) purple

Mordant I (Iodine)” basic “ purple

Decolorizer A (acetone-alcohol) colorless

Counterstain S (Safranin) red

Purpose Ziehl-Neelsen(Hot)(CAM)

Kinyoun (Cold)(CAM)

Rhodamine-Auramine(Fluorochrome)

Primary Carbol Fuchsin Carbol Fuchsin Auramine-rhodamine

Mordant Heat Phenol, Tergitol

Decolorizer 3% acid alcohol 3% acid alcohol 0.5% acid alcohol

Counterstain Methylene blue Malachite green

Quenching agent-0.5% KMNO4

Result AFO- RedNAFO-Blue

AFO-RedNAFO- Blue

AFO- YellowFluorescence

A. Staphylococci and Similar Microorganisms1. Staphylococcus aureus

a. Approximately 30% of the population carries S. aureus resident flora,

b. Isolated from abscesses, wound infections and carbuncles.

c. Causes food poisoning (via enterotoxin) pneumonia, osteomyelitis, endocarditis, wounds, staphylococcal scaldes skin syndrome,etc.

d. Produces six types of enterotoxin and toxic shock syndrome toxin-1 (TSST-1)

e. Identifying characteristics1. Gram + cocci arranged in

clusters2. Colonies are opaque and smooth.3. Catalase and coagulase positive4. Latex agglutination assay detects

clumping factor and protein A on the surface of S. aureus.

5. Negative for ability to metabolize the substrate pyrrolidonyl-alpha naphthylamide (PYR)and orthithine.

6. Staphylococci can be tolerate the high salt concentration (7.5) of mannitol salt agar (MSA).

7. Penicillin resistance is due to beta lactamase production. Methicillin resistant S. aureus (MRSA)is resistant to B-lactam antibiotics because of production of altered penicillin-binding proteins. Rare strains of vancomycin-intermediate S. aureus (VISA) and vancomycin-resistant S. aureus (VRSA) have been reported.

2. Coagulase-negative staphylococcia. common skin flora mostly nonpathogenic. this group causes UTIs and associated

with infections of catheters.b. Gram-positive cocci arranged in clustersc. Colonies appear white to gray on blood

agar and nonhemolytic.d. Catalase positive and coagulase negativee. Commonly encountered species.

1. Staphylococcus epidermidis- Most common species of coagulase negative staphylococci, novobiocin susceptible.

2. S. saprophiticus-Significant only in UTIs, resistant

3. S. lugdunensis-Frequent cause of endocarditis,ferments mannitol. PYR

positive, and typically clumps in plasma

3. Micrococcusa. Micrococcus spp. are considered normal flora of the skin and mucous membranes. they rarely cause infections.

B. Streptococcaceae and similar Microorganisms1. General Characteristics a. Catalase negative, gram-positive cocci arranged in pairs and chains.

2. Group A Streptococcus (S. pyogenes)a. Infections are spread by respiratory

secretions, and some children may carry the bacteria in the respiratory tract without illness. However,

S. pyogenes is always considered pathogenic.b. Infections caused include strep throat (pharyngitis), impetigo, cellulitis,scarlet

fever, pneumonia, otitis media and necrotizing fascitis.

c. Sequelae include rheumatic fever and post-streptococcal glomerulonephritis.

d. Susceptible to bacitracin (A disk) and PYR positive, but often identified by serological latex agglutination test.

e. Colonies are pinpoint (<1mm),translucent,and will show a large zone of beta-hemolysis.

f. Virulence factors1. M protein-inhibits phagocytosis

and antibodies to M protein are protective.2. Streptococcal pyrogenic exotoxin-

causes rash and scarlet fever.

3. Streptokonase-dissolves clots4. Streptolysin O and streptolysin S lyse erythrocytes,platelets and neutrophils.5. Hyaluronic acid- capsule inhibits phagocytosis.

3. Streptococcus dysgalactiae subsp. Equisimilisa. clinical spectrum as resembles S. pyogenesb. Lancefiel group Cor G antigens.

4. Group B Streptococcus (S. agalactiae)a. Normal flora gastrointestinal tract of

humans and animals.b. Important cause of infections in

OB/GYN patients: 25% of all females carry the bacteria as normal vaginal flora.

c.Early onset infections, neonates acquired during birth, resulting in sepsis and meningitis.

5. Group D Streptococcus a. Normal fecal and oral flora.

b. associated with wound infections, UTIs, and abdominal abscesses.

c. indicator of colon cancer if isolated in blood culture.

d. Colonies are gray to white, translucent, round and convex.

6. Viridans streptococcia. Normal flora of the oral cavity, respiratory tract, and gastrointestinal (GI) tract mucosa.b. Bacterial endocarditis in people with damage heart.c. may enter after dental procedures.

7. Streptococcus pneumoniaea. Normal upper respiratory tract flora but can cause: 1. Lobar pneumoniae in elderly and alcoholics 2. Otitis media in infants and children 3. Meningitis

8. Enterococcusa. Most commonly encountered species E. faecalis and E. Faecium.

9. Gemellaa.have been associated with a number of infections, including endocarditis, meningitis, brain abscesses, lung abscesses and osteomyelitis.

10. Leuconostoca. havebeenlinkedto ostemyelitis,ventriculitis,

postsurgical endopthalmitisbacteremia in neonates.b.vancomycin resistant.

C. Aerobic Non-Spore-Forming Gram-Positive Bacilli1. Listeria monocytogenes

a. Causes spontaneous abortion and meningitis in animanls

b. Found in environment(soil and water)and is normal flora of the vagina and intestines in humans.

c.cause of variety of infections in neonates,pregnant women and immunosuppressed patients. Meningitis is a

common outcome of infection.d.umbrella motility insemisolidroom temp,tumbling motility in wet mount.

2.Corynebacteriuma. C.diphtheriae causes diphtheria. 1. picket fences or chinese letters

can be pleomorphic.b. C. jeikeium causes mosocomial

infections after prosthetic implants and infections in immunocompromised patients.

c.C. urelyticum1. cause UTIs2.urease positive

D. Aerobic spore-Forming Gram-Positive Bacilli1.Bacillus anthracis-anthraxa.Three clinical forms:1. Cutaneous anthrax-black eschars2. Pulmonary anthrax- Wool-sorter’s disease3. Gastrointestinal anthrax-ingestions of spores.

2.Bacillus cereusa.food poisoningb.B. cereus and B. subtilis-common lab. Contaminants.

A.Neisseria gonorrhoeae1. In males-causes acute urethritiscontai