UAMS Microbiology Module 1

Who made the Gram stain?

Christian Gram

What are the reagents for the Gram stain?

Crystal violet (initial stain placed on bacteria, purple or dark blue look)

Iodine (will mix with the crystal violet present in bacteria to help it stick, makes mordant).

Acetone-alcohol decolorizer (usually 1:1, ethanol 95%:acetone)

Safranin (secondary stain, the pink one).

How do you make slides from a colony on an agar/media

Mix with a drop of saline.

Generally, place the drop if saline on the slide and then take a loopful of bacteria from a colony and swirl it around.

What are the methods to fix slides?

Just think heat.

Generally with a slide warmer at 60C

Pass through flame 2-3 times.

Hold at front of incinerator (this part gets really hot so use tweezers).

Flood smear with methanol and

What can happen due to the cells themselves, and not the process of staining?

Some GP organisms, as they age or from antibiotics, will have weaker cell walls and may appear GN as they can’t hold in the stain.

The best results will be on <24hr old cultures from non-inhibitory media (blood agar, since it’s only nutrients).

Overheating may distort some cells as well.

What are the characteristics of agar media?

Agar media: Polysaccharide melts at 100 C; stays liquid until 45-50C -Media may be formed into plates, slants, or deeps.

• Semi-solid: 0.3-0.5 % agar

• Solid: 1-2% agar

What does the streak plate method do?

It’s the 3-4 quadrant streaking on the plates.

Separates & semi-quantitatively enumerates
organisms present in patient specimens or mixed cultures.

Principle: Organisms are separated by spreading them over the
surface of the agar plate.

Each successive section has fewer organisms (section 3 < section 2 < section 1) & organisms farther apart.

Colony-forming-units (CFU): Individual or small groups of cells which multiply to form visible colonies. Isolated colonies appear after incubation.
May be unnecessary to sterilize loop between sections when working with patient specimens.

Interpretation:

• Growth only in the first section = 1+ = Few, sparse, or light

• Growth in the first and second sections = 2+ = Moderate.

• Growth into the third section = 3+ = Many or heavy.

What are the types of media?

Nutrient: grows most kinds of fastidious organisms.

Enriched: Supplemented with various substances (blood, blood by-products, vitamins, yeast extract, carbohydrates, etc.). Grow fastidious organisms (special nutritional requirements).

Enrichment broths: Enhance growth of certain MO while inhibiting other organisms.

Selective: Have antimicrobial substances (e.g., dyes, antibiotics, &
chemicals). Inhibit some microbes while allowing the growth of others.

Non-selective/inhibitory: No antimicrobial substances & allow growth of many organisms.

Differential: Metabolic differences distinguish microorganisms. Identification media are differential.

Nutrient media examples

Nutrient agar

trypticase soy broth

Enriched media examples

Sheep blood agar plate (BAP): Most commonly used medium, Grows many microorganisms

• Sheep BAP: Nutrient base (e.g., trypticase soy
  agar) supplemented with 5% sheep blood.

• Blood agar made with human, rabbit, or horse
  blood is used in some situations
  

Chocolate agar (CHOC): Grows many organisms including fastidious bacteria such as Haemophilus.

• Modified blood agar; made by heating sheep RBC
  to release nutrients or by adding nutritional
  supplements to a nutrient agar base

Enrichment broth examples

Gram-Negative (GN) broth & Selenite broth: Used to isolate Salmonella and Shigella from fecal specimens.

LIM broth & Trans-Vagil broth Used to isolate Group B
Streptococci from vaginal/rectal specimens.

Selective broth examples

Have antimicrobial substances (e.g., dyes, antibiotics, & chemicals). Inhibit some microbes while allowing the growth of others.

• PEA agar is blood agar supplemented with phenylethyl
  alcohol.

• CNA agar is blood agar supplemented with the antibiotics
  colistin and nalidixic acid.

• Gram-positive organisms grow on PEA agar and CNA agar while most gram-negative bacilli do not. (Anaerobic gram-negative bacilli grow on anaerobic formulation of PEA).

Non-selective media examples

No antimicrobial substances & allow growth of many organisms.
Examples: BAP and CHOC

Some medias can be multiple. BAP is both nonselective, enriched, and differential due to growing anything, differing by hemolysis, and containing blood cells.

Differential media examples

Metabolic differences distinguish microorganisms.

• Identification media are differential.

• Examples: Carbohydrate fermentation media & enteric media.

What is the most common temp used in micro?

35C (though the range is 35-37C)

Other temps are used for specific pathogens, there will either be certain incubators for this or one incubator will be used and the temp changed depending on the organisms that need recovering.

What are the other few temps needed for micro?

Yersinia enterolitica is 22-25C

Campylobacter jejuni is 42C

What are the types of atmosphere conditions needed?

Aerobic: Air approximately 21% oxygen. Obligate aerobes require oxygen.

Anaerobic: No oxygen, O2 toxic for strict anaerobes, some anaerobes tolerate low O2 level.

Facultative anaerobes: Grow both aerobically and anaerobically.

Microaerobic (also known as microaerophilic): Organisms require a decreased level of oxygen (5% -10%).

Capnophilic organisms: require an increased level of CO2 (5% to 10%).

• Special CO2 incubators

• Candle extinction jars: Place culture plates & lighted, white candle in jar and seal. Colored or scented candles may be toxic. Before the candle burns out, the flame will use some of the oxygen to produce an atmosphere of 1% to 3% CO2.

Humidophilic microbes: require increased humidity (70% to 80%). Some incubators have built-in humidifying systems or water-filled pans to increase humidity.

*The difference between microaerobic and capnophilic organisms is very small. Just remember the key words decreased and increased. (imagine one coming from the bottom to get increased oxy, and the other going down to get decreased oxy).

What are some other considerations for incubating specimens?

Keep lids lightly screwed on unless stated.

Keep plates upside down (agar in the air) so condensation doesn’t drip on the media and colonies.

What is hemolysins and what are the types produced?

It is damage to the RBCs in the agar.

Gamma: No clearing at all.

Alpha hemolysis: green apple, partial clearing of the agar.

Beta: Full clearing, should be able to see through it.

Another called Alpha Prime is alpha hemolysis surrounded by a zone of beta hemolysis (partial clearing and full clearing around that)

What are ways to disinfect the Micro lab?

Sterilization: Destroys all MO including bacterial spores. Exception: Prions (agents that cause transmissible spongiform encephalopathy). Require special methods to destroy.

Disinfection: Pathogenic organisms eliminated. Viable bacterial spores may be present. Antiseptic: Disinfectant used on living tissue (skin)

3) Physical methods: Usually sterilize

• Autoclaves: Pressure & steam sterilize at 15 pounds per square inch (psi) with Temperature of 121 C. 15 min exposure time sufficient for most items. 30-60 mins for infectious waste

• Dry heat: 160 - 180 C for 1.5 to 3.0 hours. Sterilizes glassware/items not tolerating moist heat

• Filtration: Liquids, air, and other gases can be filtered.

• Radiation (Ionizing & ultraviolet radiation.

Chemical agents: Usually disinfect:

• Alcohols: Ethyl or isopropyl alcohol (60 - 90%)

• Aldehydes: Glutaraldehyde

• Phenolics (chemically modified phenols)

• Quaternary ammonium compounds (“quats”)

• Halogens: Bleach (has chlorine, Sodium hypochlorite (NaClO)). 1:10 dilution recommended for blood spills. Clean area first then disinfect (chlorine
  inactivated by organic matter)

• Iodophores (Disinfectants & antiseptics) Iodine compounds/Povidone-iodine.

What are the different biosafety levels?

Level 1: won’t cause disease in normal healthy adults. Wash hands and wipe down things.

Level 2: routinely used by most labs. Human pathogens not transmitted by inhalation. Coats, limited access, and sharps.

Enterics, Herpes simplex virus, Staph. aureus

Level 3: MO transmitted by inhalation, physically separated area, possible respirators, negative pressure, (our labs level).

Level 4: Extremely contagious and dangerous organisms (Ebola).

Mutualism

Both the bacteria and human benefit.

Like intestinal bacteria, they help digest food and make some Vit K for us.

Commensalism

Human is not affected while bacteria benefits.

Normal flora is often this.

Parasitism

Bacteria benefit at the expense of the human host.

Like Grp A strep.

Opportunistic pathogens

Not normally a pathogen unless the body is weakened (immunoompromised, wounds).

Virulence

Due to a variety of microbial factors (toxins, capsules, enzymes). Virulent microorganisms are more likely to cause disease

Carrier

Will carry the disease and can give it to another.

Usually asymptomatic.

Zoonosis

Animal disease in a human.

What are the human defense mechanisms?

Intact skin and mucous membranes

Mucous membrane cleansing activity: Mucus & cilia

Production of antimicrobial substances

• Lysozyme: Enzyme disrupts cell walls of gram-positives;
  found in tears, saliva, and other body secretions.

• Fatty acids: Produced by sebaceous glands; reduces skin’s pH, which inhibits microbe growth

• Antibodies on mucous membranes. Phagocytosis by PMNs, macrophages, and monocytes

• Host susceptibility factors: Nutritional status, age, stress, genetic makeup, and preexisting conditions (surgery, foreign bodies, etc.).

What happens in the invasion of a body by bacteria?

Attachment to host cells:

• Some bacteria (N. gonorrhoeae use pili (short, filamentous, surface structures) to adhere to host.

• Proliferation: Organisms grow and multiple if they effectively compete with the host and other microbes for nutrients.

Evasion of host defenses:

• IgA proteases produced by some organisms & destroy IgA

• Inhibition of Phagocytosis by Capsule: Protein/polysaccharide coats bacterial cell
  (i) Phagocytes engulf encapsulated MO only
  when specific anticapsule antibodies
  present. (Example: Streptococcus pneumoniae)

• Some microbes ingested but not killed by
  phagocyte.

• Antigenic variation: Some MO (Borrelia) change their
  surface antigens as host responds to MO. Host has to play immunologic “catch-up”.

What kinds of toxins are made by bacteria?

Exotoxins: Extracellular proteins produced by metabolizing bacteria.

• Harm host cells by damaging the membrane or by
  interfering with normal metabolic functions.

• Toxins may be named for their site of action.

• Neurotoxins - nervous system.

• Enterotoxins - gastrointestinal tract.

Endotoxins: Lipopolysaccharide (LPS) part of gram-negative cell wall.

• LPS composed of carbohydrates and lipid A (toxic part).

• Once bacteria die, the toxin is released.

• Can cause fever, shock, bleeding, and death.

How do normal flora help the human body?

Can interfere with pathogens by:

• Producing bacteriocins (antimicrobial substances).

• Reducing a body site’s pH (e.g. lactobacilli in female genital tract).

• Competing for nutrients.

NF of Gastro tract

Anaerobic bacilli (Bacteroides, Clostridium)

Enterococci
Anaerobic cocci

Staphylococcus aureus

Gram-negative enteric bacilli

Yeast (Candida)

Genitourinary NF

Lactobacilli

Diphtheroids

Anaerobic bacilli (Bacteroides, Clostridium)

Streptococci

Anaerobic cocci

Gram-negative enteric bacilli
Staphylococci (S. epidermidis, S. aureus)

Acinetobacter

Enterococci

Yeast (Candida)

Respiratory Tract NF

Staphylococci (S. epidermidis, S. aureus)

Haemophilus

Streptococci (viridans, pneumococci)

Gram-negative bacilli (enterics, nonfermenters)
Enterococci

Anaerobes

Diphtheroids

Spirochetes
Neisseria

Yeast (Candida)

Skin NF

Staphylococci (S. epidermidis, S. aureus)

Diphtheroids

Micrococci

Gram-negative bacilli (enterics, nonfermenters)
Streptococci (nonhemolytic)

Anaerobes

Enterococci

Yeasts and fungi

Staphylococcus characteristics

Staphylococcaceae (family): Staphylococcus (genus)

Very common NF, will cause serious diseases.

Two groups:

• Coag Negative Staph. Since theres so many, you generally just say coag neg staph until further ID.

• Coag Positive Staph: Staph. aureus.

Growth conditions: Faculative anaerobic, will grow on routine media (BAP, CHOC, CNA, PEA).

Normally in anterior nares, mucus membranes and skin.

Staph aureus Characterisitcs (think butter)

Coagulase +

Catalase +

GS: grape clusters.

BAP: Opaque, buttery/smooth, with beta hemolysis. Slight yellow color.

MSA: Yellow, yellow zones. (it fermented the mannitol and produced acid, turning the indicator yellow).

Coag Negative Staph. characteristics (think margarine I guess)

Catalase +

Coagulase -

BAP: opaque white butter, gamma hemolysis (none).

MSA: Red, red zones (doesn’t ferment mannitol).

Diseases caused by Staph. aureus

Invasive diseases
(1) Skin infections (e.g., folliculitis, furuncles, impetigo, and
carbuncles)
(2) Abscesses, wound infections, osteomyelitis, + others
(3) Antibiotic-resistance common.

Toxigenic diseases:

• Staphylococcal food poisoning: Improperly stored food contaminated with enterotoxin-producing staphylococci. Symptoms: Diarrhea and vomiting within hours.

• Scalded skin syndrome: Usually occurring in neonates and infants due to exfolitin toxins: Skin sloughs.

• Toxic shock syndrome (TSS): Toxic Shock Syndrome Toxin - 1 (TSST-1). Symptoms: high fever, hypotension, confusion, diffuse rash, and acute renal failure. Most early cases were associated with menstruating women using hyper absorbable tampons. TSS occurs in men and women as a complication of a number of staph infections.

Diseases caused by Coag-negative Staph.

Once considered to be nonpathogenic
Most infections in Immunocompromised patients, those with indwelling medical devices (catheters).

S. epidermidis: Most commonly isolated species, especially from
cases of prosthetic valve endocarditis.

S. saprophyticus: Urinary tract infections, especially in young,
sexually active women.

S. lugdunensis: Can cause an aggressive form of endocarditis.
important to differentiate from other STPCN because of different
interpretive criteria for oxacillin.

Catalase test principle

Catalase (enzyme) breaks down hydrogen peroxide
to water and oxygen with bubbles formed by the released oxygen.
catalase
H2O2 ⎯⎯⎯⎯→ H2O + O2 (bubbles)

Procedure: Place colony of test organism onto a glass microscope slide. Add a drop of 3% H2O2.

Interpretation:

• Positive: *Immediate production of bubbles.

• Negative: No bubbles.
  

False-positive reactions:

• If test performed in reverse order with an iron or nichrome
  loop.

• Carefully remove colonies growing on BAP (RBC have small amount of catalase).

• *Pseudocatalase: Some bacteria (e.g., enterococci)
  produce pseudocatalases which decompose H2O2. These false-positive results may be avoided by reading the test within 20 to 30 seconds.

Coagulase test principle.

Coagulase converts fibrinogen > fibrin

The reagent is just plasma containing coagulase.

There are a few different versions: the slide and tube coagulase tests.

Slide coagulase (bound coagulase, clumping factor) test Principle: Bound coagulase attached to the cell wall (hence the name). When S. aureus is mixed with plasma, visible clumps of cells appear as fibrin strands are formed.
Procedure: Use microscope slide

• Mix organism in saline - heavy smooth suspension

• Mix in a drop of plasma with applicator stick

Interpretation:

• Positive: Appearance of clumps in 10 to 15 seconds. Report organism as S. aureus.

• Negative: No clumping.

• Invalid: MO autoagglutinates (clumps in saline).

• Perform tube test: On slide coagulase-negative isolates since not all S.aureus strains are slide coagulase positive. And on strains which autoagglutinate

Tube coagulase test Principle: Extracellular (free) coagulase forms complex with coagulase-reacting factor (CRF), in plasma.

• Complex converts fibrinogen to fibrin.

• Procedure: Test organism emulsified in plasma in a tube. Tube incubated at 35 C for four hours.

• Interpretation: Gently tilt tube to detect clot formation (+ result). Reexamine negative tubes after overnight
  incubation at room temperature.
  

Sources of error:

• Read slide test within 10 secs to avoid false-(+) results.

• False (+) or false (=) results with nonsterile plasma

• False-negative tube tests may occur with S. aureus strains that lyse clot after prolonged incubation.

• Methicillin-Resistant S. aureus (MRSA): Often slide coagulase negative and tube coagulase positive

• Colonies from media with a high salt content (MSA) more
  likely to autoagglutinate. Best to use colonies from BAP.

Latex agglutination Test principle

Latex agglutination (LA)test.

Many clinical laboratories replaced slide and tube coagulase tests
with commercial LA test kits.

Detects clumping factor, protein A (a S. aureus cell wall antigen), and possibly capsular polysaccharides.

False (=): possible with MRSA.

False (+): possible with other Staphylococcus/Micrococcus species.

Passive hemagglutination/fluorogenic methods also available

Novobiocin susceptibility test principle

Used to presumptively ID S. saprophyticus in urine cx.

Principle: Agar plate inoculated for confluent growth with test MO. Paper disk with novobiocin (an antibiotic) placed on agar. Novobiocin diffuses into the agar and produces a gradient
of changing drug concentrations. If test organism inhibited by drug, a zone of no growth (zone of inhibition) occurs around the disk.

Procedure:

• Make broth suspension (0.5 McFarland turbidity standard).

• Test MO swabbed onto agar plate (BAP or Mueller-Hinton).

• Place paper disk with 5-g novobiocin onto inoculated
  area.

• Incubate plate incubated for 18-24 hours at 35 C.
  

Interpretation:

• Susceptible: Zone of inhibition > 16 mm in diameter.

• Resistant: Zone < 16 mm (presumptive S. saprophyticus).

• Source of error: Rarely other staph species resistant.

Ornithine decarboxylase test principle

Perform on coag-neg (tube coagulase) staphylococci isolated from blood cultures; presumptively ID S. lugdunensis.

Principle: Checks if MO can degrade ornithine (amino acid).

Decarboxylases cut carboxyl (COOH) from amino acids → amines.

Amines make environment alkaline

pH indicators detect pH change

Procedure:

• Inoculate organism to Moeller’s broth with ornithine.

• Control tube (containing Moeller’s base medium) is
  inoculated.

• Cover each broth with sterile mineral oil (produces
  anaerobic conditions).

• Incubate tubes.

Interpretation:

• Positive: Amino acid tube = purple & control tube = yellow (presumptively S. lugdunensis).

• Negative: Yellow in both tubes.

What are some other ID tests?

Automation (PCR) with rapid multi test systems.

Used for genetics and getting the ID down to the species.

Micrococcus Characteristics

Catalase +

Coagulase -

Large GPC (often tetrads/four).

Media growth: lemon yellow color common.

Very similar to Staph (skin, mucus membranes, environment).

Will rarely cause disease, normal contaminant.

Bacitracin: S

Mod Oxidase test principle

Detects cytochrome oxidase.

Enzyme oxidizes tetramethyl--phenylenediamine (oxidase
reagent) to form indophenol.

Modified by adding dimethyl sulfoxide (DMSO) which makes
bacterial cells more permeable to the oxidase reagent.

Perform by rubbing test MO onto paper disk with reagent.

Positive: Blue color within 30 seconds

Rothia (Stomatococcus) mucilaginosus characteristics

Normally in oral cavity.

GPC that form clumps and tetrads. Is encapsulated.

Catalase -

Media growth: sticky staph (strongly adheres to agar surface), gray-white mucoid.

Is an opportunist, not very commonly seen.

Cheat sheets for Staphs

Big cheat sheet.

Flow chart of staphs

Streptococci characteristics

GPC in pairs and chains

Growth conditions: Faculative anaerobe.

Catalase -

What is the main way to ID the streps?

Serogrouping: helps identify many strep (but not all):

• Based on C carbohydrate in cell wall (Lancefield grouping).

• Latex agglutination, EIA or coagglutination technology.

• Human serogroups: A, B, C, D, F, & G.

• May have multiple organisms in same group, thus additional testing may be necessary for clinically significant isolates.
  

Physiologic properties:

• Biochemical reactions

• Organism given a genus and species name.
  

Hemolytic activity: gamma, alpha, beta.

Common names (lab jargon): beta strep, alpha strep, and pneumococci.

Strep pyogenes (Grp A strep)

aka GAS

Hemolysis: Beta (fullc learing). Stab the plate to see this more clearly.

Toxins:

• Streptolysin S: oxygen stable,

• Streptolysin O: oxygen labile (needs no oxy or won’t be seen)

• This is why all of the suspected strep plates are put in the CO2 incubator.

Colony morpho: Small, translucent, large beta hemo.

No normal habitat.

A disk S: (once this is known you can prelim it as GAS).

SXT disk: R

PYR:

Think of how a forest fire can clear a lot of land for the hemolysis.

Diseases:

Suppurative (pus forming) and toxic diseases:

• Pharyngitis (“strep throat”), scarlet fever, bacteremia.

• Skin infections/pyoderma (e.g., erysipelas, cellulitis & impetigo).

• Streptococcal toxic shock syndrome (tsmpons).

Nonsuppurative sequelae:

• Rheumatic fever.

• Acute Glomerulonephritis.

When testing is done for detection, always verify negative results with a culture.

What are the Beta hemolytic streps?

S. pyogenes,

S. agalactiae

Grps C, F, G

Bacitracin (A disk) test priniciple

(“A” disk identifies group A strep)

Principle: Low concentration bacitracin (antibiotic) inhibits GAS

Procedure:

• Inoculate BAP for confluent growth.

• Paper disk (0.04 units of bacitracin) placed onto plate.

• Incubate overnight..
  

Interpretation: Susuceptible (S) = Any zone of inhibition

Reporting results: Bacitracin susceptible -hemolytic strep may be reported as “Presumptive group A streptococci”.

Sources of error (SOE):

• Test only Beta-hemolytic strep; some alpha-hemolytic strep (S).

• False (+) with some group B, C, & G streptococci.

• GAS rarely bacitracin resistant.

• Many labs have replaced the A disk with PYR & serogroup antigen tests.

Trimethoprim-sulfamethoxazole (SXT) disk susceptibility test principle

Principle:

• Groups A and B streptococci (R).

• Groups C, F, and G are (S).

• Some labs use media containing SXT to select for GAS.

Procedure: Same as A disk

Interpretation: S = any zone

PYR (L-pyrrolidonyl--naphthylamide) hydrolysis test principle

Principle
L-pyroglutamyl aminopeptidase
L-pyrrolidonyl--naphthylamide ⎯⎯⎯⎯⎯⎯→ Free -naphthylamine

Free -naphthylamine + N,N dimethylaminocinnamaldehyde → Red color

Procedure:

• Place test MO onto disk with PYR; wait a few minutes.

• Add developing reagent.

Interpretation:

• Positive: Red.

• Negative: No color change.
  

Source of error:

• Variety of other MO PYR (+).

• Interpret in conjunction with MO’s gram stain, colonial morphology, type of hemolysis, & catalase reaction.

S. agalatiae (Grp B strep).Think of clouds with the hemolysis being the rain directly underneath.

Hemolysis: Beta, some gamma. The hemo is normally directly indernesth the MO.

Colony morpho: small, flat, cramy white.

NF of genitourinary tract.

Very dangerous in babies (can cause meningitis/sepsis). This is why the GBS is done in prenatal care so it can be treated.

A disk: R

SXT: R

PYR: +

How is the GBS screening test done?

Use enrichment broth:

• LIM Broth (Todd Hewitt broth w/ colistin & nalidixic acid)

• Trans-Vag Broth (Todd Hewitt broth w/ gentamicin & nalidixic acid + the addition of 5% defibrinated sheep blood).

• If growth, subculture to BAP at 24 hrs; hold broth for 48 hours.

CAMP test principle

Principle:

• Some S. aureus produce “beta-lysin” (a toxin).

• GBS produce “CAMP factor”.

• CAMP factor + beta-lysin = arrowhead-shaped area of
  enhanced hemolysis.

Procedure:

• Streak S. aureus down the middle of a BAP.

• Streak test MO perpendicular to staph streak, incubate.

Interpretation:

• Positive: Arrowhead-shaped hemolysis (GBS).

• Negative: No enhancement (Most other streptococci).

Sources of error:

• False (+): Some GAS (10%) CAMP positive.

• False (=): Approximately 2% of GBS CAMP negative.

• Not all S. aureus strains produce beta-lysin.

• Run (+) & (=) controls each time test performed.
  

Hippurate Hydrolysis test principle

Principle: Can detect the glycine and sodium benzoate.

hippuricase hippurate ⎯⎯⎯⎯⎯→ sodium benzoate + glycine

Sodium benzoate detection: Special broth + MO incubated overnight
(2) Test supernatant by adding 7% ferric chloride
(3) Positive: Precipitate after 10 minutes
(4) Negative: Precipitate clears within 10 minutes

sodium benzoate + 7% Ferric chloride ⎯⎯⎯→ precipitate.

Glycine detection:

• Place disk with hippurate in tube with water.

• Add test MO, incubate for 2 hours, add ninhydrin.

• Positive : Blue/purple color after 10 mins.

• Negative: No color change
  glycine + ninhydrin ⎯⎯⎯→ blue/purple color.
  

Sources of error (SOE):

• Sodium benzoate method: Reading too soon.

• Ninhydrin method: Wrong media

• Other MO hippurate (+); Interpret in conjunction with MO’s gram stain, catalase, & colonial morphologly.

Grp C, F, and G streptococci

Made of several species.

Hemolysis: Beta

NF: Resp, gastro, vag.

Many diseases (Grp C + G can cause pharyngitis).

Grp D streptococci (

Hemolysis: Alpha, gamma.

NF in gastrio.

Split into two groups: Enteroccus and non-enterococcal grp d strep.

Enterococcus is much more resistant to antibiotics.

S. gallolyticus (previously s.bovis) found in humans. (is non-enterococcal GDS). Is associated with colon cancer.

Bile-esculin test principle

Principle: Determines ability to hydrolyze esculin if 40% bile present. High bile concentration inhibits many organisms

Agar has ferric ions:
esculin ⎯40% bile→ esculetin + glucose
esculetin + ferric ions ⎯⎯→ black color

Procedure: Inoculate agar (tube or plate) & incubate
Interpretation:

• Positive: Black (Nonenterococcal GDS & enterococci)

• Negative: No color change

SOE: some viridans strep > faint black color if heavy inoculum.

6.5% bile esculin test principle

Principle: Determines MO’s ability to grow in 6.5% NaCl (Some manufacturer’s include pH indicator).

Procedure: Inoculate broth tube with 6.5% Na Cl, incubate.

Interpretation:

• Positive: Turbidity or acid pH.

• Negative: No growth or pH change.

Enterococci: Bile-esculin (+); 6.5% salt (+)
*Nonenterococcal GDS: Bile-esculin (+), 6.5% salt (=).

Source of Error:

• Some other gram (+) MO may grow in 6.5% salt.

• Do bile-esculin test at same time as the 6.5% salt test.

Enterococcus

E. faecalis and E. faecium are most common isolates.

Hemolysis: gamma or alpha, rare beta.

Colony morpho: small grey, shiny.

NF in oral/gastro tract.

Disease: variety, antimicrobial R is a big concern.

Catalase -

Bile esculin + 6.5% salt +

PYR hydrolysis +

S. pneumoniae

Pneumococcus

GS: lancet shaped GPC in pairs, kinda like 2 footballs.

Growth needs: CO2 optimal.

Hemolysis: Alpha.

Colony morpho:

• Mucoid: Large amounts of polysaccharide capsule (> 80 types).

• Umbilicated: Depressed center w/ raised edges, indented.

• Due to autolytic enzymes.

• Young colonies = small mounds. Depressed center as age.

NF in upper resp tract.

Most common cause of community acquired bacterial pneumonia. Other variety of diseases (meningitis, pneumonia, otitis media).

Used to ID with the capsule.

Optichin S test principle

P disk (P disk is used to identify pneumococci).

Principle: Ethylhydrocupreine hydrochloride (optochin) inhibits pneumococci.

Procedure: Same as A disk; incubate in CO2 overnight.

Interpretation:

• Zone depends on disk size (6 mm or 10 mm).

• Susceptible: Zone > cutoff size.

• Resistant: No zone.
  

Questionable results:

• Zone present but < cutoff size

• Do bile solubility test

• P disk (S) alpha-hemolytic strep = “Presumptive pneumococci”

Bile solubility test principle

Principle: Bile salts (sodium deoxycholate and sodium taurocholate) accelerate autolysis.

Broth method:

• Turbid suspension of MO prepared in broth or saline.

• Divide equally into 2 tubes; Add bile to 1 tube.

• Incubate both tubes at 35 C for 2-3 hours.

• Positive: Clearing (lysis of the cells) S. pneumoniae (+).

• Negative: Turbidity (no lysis).

Plate method:

• Add bile to colonies on BAP; incubate.

• Positive: Lysed colonies (pneumococci).

• Negative: Intact colonies.

• Sources of error:
  (1) Mixed cultures.
  (2) Nonpneumococcal colonies may be lifted off the plate.

Quelleng reaction test principle

Principle:

• IDs isolate as pneumococcus & determines its capsular type.

• Specific capsular ABY + encapsulated cells → ABY-AGN
  reaction - gives appearance of swelling (optical illusion).

• No “swelling” if ABY does not match capsular antigen.

• Polyvalent ABYs react with many capsule types.

Procedure:

• Capsule antibody + suspension of pneumococci.

• Second (control) suspension is left untreated.

• Examine wet mounts of each suspension.

Interpretation:

• Positive
  (a) “Capsular swelling” in ABY treated cells and
  (b) No “swelling” in the untreated cells.

• Negative: No “swelling”

Strep. viridans

several species

hemolysis: alpha or gamma.

NF in Resp tract, gastro, skin.

Diseases: vary, endocarditis in pts with damaged/artificial heart valves, called subacute bacterial endocarditis.

Usually ID by rule out.

Alpha or gamma-hemolytic strep = viridans streptococci if they are NOT enterococci,
GDS not enterococci, pneumococci, or group B streptococci.

Strep anginosus

Formerly S. milleri.

Included with 3 names: S. anginosis, S. constellatus, & S. intermedius.

Hemolysis: all of them. Will be minute colonies.

Microaerophilic, use increased C02.

NF in oral cavitiy, gastro, vag.

Satelliting streptococci

Other names: Nutritionally deficient, Thiol requiring, Nutritionally variant, Pyridoxal dependent, Vitamin B6 dependent, Symbiotic streptococci.

Resemble viridans streptococci, but genetically distinct.

Current terminology: Abiotrophia and Granulicatella.

NF in Upper resp, urogenital, gastrointestinal tract.

Diseases: Endocarditis + others.

How are Satelliting strep grown?

Nutritional requirements:

Require vitamin B6 (pyridoxal)
b) Will not routinely grow on BAP or chocolate agar (CHOC)
c) Can add pyridoxal to growth medium or
d) Can be supplied by another organism growing on the agar plate

Satellite procedure
a) “Deficient” organism spread onto the surface of an agar plate.
b) Single streak of 2nd organism (usually a staph) made through area
c) Incubate overnight incubation CO2
d) Faint growth or tiny colonies near the “staph streak”
e) Staph makes excess nutrients which allow “deficient” strep to grow.

Clinical specimens: Mixed cultures; tiny colonies near big colonies'.

• Haemophilus: GNB

• Satelliting strep: GPC

Blood cultures: BC media contains pyridoxal & allows satelling strep to grow.

Perform “staph streak” procedure when:

• Blood culture broth shows GPC &

• No growth when subcultured onto BAP or CHOC.

Identification:

• Report of “satelliting streptococci” is usually sufficient.

• Complete identification by reference labs.

• Differentiate from other strep because they may be more R to penicillin.

What are the streptococcus like spp.?

Genera:

• Aerococcus

• Gemella

• Lactococcus

• Pediococcus

• Leuconostoc

Resemble viridans streptococci and enterococci.

Hemolysis: Alpha or gamma

NF: Human mucous membranes and the environment.

Diseases: Rare, but variety do occur

Catalase =, but a few weak +/

Vancomyocin R: Pediococcus, leuconostoc.

Vancomyocin S: Aerococcus, Gemella, Lactococcus.

There are Nucleic acid tests for further ID of them.

What are the most commonly isolated streps?

GAS

GBS

Grps C, F, G

Enterococci

GDS

Pneumococci

Viridans strep

What are the alpha hemolytic strep? (or the most likely Bacteria) And what are the tests to ID?

Most likely organisms are:

• Enterococci

• Group D streptococci

• Pneumococci

• Viridans streptococci
  

Appropriate tests include:

• P disk or bile solubility

• Bile-esculin hydrolysis

• 6.5% salt tolerance

• PYR

What is the most likely bacteria and what test would you perform for Beta hemolytic strep?

Most likely organisms are:

• GAS

• GBS

• Groups C, F, or G streptococci

Appropriate tests include:

• A and SXT disks

• PYR

• CAMP or hippurate hydrolysis

• Serogrouping

What is the most likely bacteria and what test would you perform for Gamma hemolytic strep?

Most likely organisms are:

• GBS

• Enterococci

• Group D streptococci

• Viridans streptococci

Appropriate tests include:

• CAMP or hippurate hydrolysis

• Bile-esculin hydrolysis

• 6.5% salt tolerance

• PYR

Bacillus spp. characteristics.

Spores are heat and chemical resistent.

Gram positive rods/bacilli

The spores don’t stain in the GS, will appear as holes in cells or refractile.

Culture conditions: Aerobic, faculative anaerobics.

Media: BAP, possible PEA.

Bacillus anthracis and cereus are most important.

Bacillus anthracis

Found in soil, plant material. Spores viable for decades.

GS: large gram positive bacilli. Won’t normslly see spores in direct ID from specimen.

Hemolysis: gamma

Motility: nonmotile

Catalase: positive.

Colony: rapid aerobic growth on BAP.

• Medusa head (filamentous growths).

• Ground glass look.

• Tenacious-look like egg whites when lifting edge of colony and it stays upright.

• String of pearls (chains of spherical baccilli), forms when incubated for several hours on agar with penecilli.

Must be on alert for B. anthracis with healthy pts that rapidly decline with resp illness or cutaneous ulcer.

If presumptive ID of B. anthracis, must notify public health staff and be worked up to isolation and full ID.

Bacillus anthracis diseases

Anthrax usually in herbivores. Human infection rare in U. S. Associated with imported animal products (wool, hair, or hides).

Cutaneous: most common form; black eschar skin lesion

Two other clinical forms are inhalation & gastrointestinal

Specimens: Skin lesions (eschar), sputum, feces, oropharyngeal ulcer,
CSF, and blood.

Laboratory hazard:

• Wear PPE (gowns and gloves).

• Work in a biosafety cabinet.

• Decontaminate work areas.

• BSL-3 recommended for activities w/ high potential for aerosol
  or droplet production.

What is the laboratory response network?

Consortium & partnership of laboratories
b) Provide immediate & sustained laboratory testing &
commununication in support of public health emergencies
c) Particularly in response to acts of bioterrorism

LRN laboratories are designed at sentinel, reference & national:

• Sentinel labs: Function at the local front line (e.g.,
  hospitals); provide “presumptive identication – recognize,
  rule out and refer”.

• Reference labs: Includes local local & state public health;
  provide confirmatory testing.

• National labs are Federal labs that have BSL-4
  containment facilities such as the CDC; work w/ agents
  such as Ebola, smallpox virus, etc.; provide definitive
  characterization.

Packaging critical biologic agents: Requires “triple” packaging:

• primary receptacle.

• water tight secondary packaging.

• durable outer packaging.

• Appropriate labeling of the package.

• Documentation of hazardous contents.

NAAT ID also available.

Bacillus spp (other than B. anthracis)

Found in the environment.

Usually a infectious oppurtunistic.

B. cereus causes food poisining and tissue infections (from toxins).

GS: large boxy GPR.

Colony: large irregular flat, beta-hemolytic.

Spore forming under aerobic conditions, must use biochemical tests for spp. ID.

Hemolysis: Beta

Motility: motile

Catalase: variable.

When will you test a gram negative rod for actually being a gram positive rod.

Some strains confused with nonfermentative GNR:

• Stain gram-negative.

• Colony morphology not typical.

• Spores may not be present.

• Some grow slightly on EMB or MAC.

Suspect Bacillus when a “GNR” :

• does not fit ID schemes

• Encourage spore formation by extended incubation

• Vancomycin susceptibility
  (a) Most Bacillus (S).
  (b) Most gram-negative nonfermenters (R).

Corynebacterium spp.

GS:

• GP non-spore-forming pleomorphic rods (tapered, curved, club shaped. coryne is greek for club).

• Chinese letters (V, L, Y, palisades).

• Diptheroids or coryneforms: includes Corynebacterium, Aacronobacterium, Rothia.

Note: “corynebacteria” refers to the genus Corynebacterium; “coryneform” refers to
irregularly shaped GPR. Corynebacterium one of several coryneforms.

Growth conditions: aerobic, faculative anaerobics. small colonies on bap at 35C.

NF: Skin, resp tract, GI.

Non-motile.

Catalase: +

Corynebacterium diptheriae diseases

Disease: Diptheria.

Usually infects upper respiratory tract; sometime skin. Has toxigenic strains (toxin produced).

Infected with beta-phage (a virus) which has toxin gene. Causes upper respiratory tract and systemic damage.

Pseudomembrane may block airway.

Nontoxigenic: Pharyngitis or a less severe diphtheria.

Prevent through immunization with toxoid.

Specimens: Throat, nasopharyngeal, and wound material.

Corynebacterium diptheriae

Has typical coryneform morphology, is not specific for C. diptheriae.

Can do the methlyene blue stain: Stain smears from specimens or cultured MO

• Observe for metachromatic granules.

• Reddish purple to deep blue, intracellular granules.

• May be in other Corynebacterium species.

Media: BAP, tellurite, Loeffler/Pai agars.

Elek test: positive for the toxin.

What are the agars used for C. diptheriae and why?

BAP: General purpose, nonselective medium grows C. diphtheriae, Group A, C & G strep & Arcanobacterium.

Tellurite media: Cystine-tellurite medium and Tinsdale agar.

• Contain tellurite; Selective and differential.

• Tellurite inhibits many normal flora MO.

• Most corynebacteria not inhibited; gray to black colonies.

• Other MO can also make black colonies.

• Tinsdale: C. diphtheriae: Black colonies with brown halo. A few other Corynebacteria also have this colony.
  

Loeffler medium:

• Serum + other factors.

• Promotes C. diphtheriae growth & metachromatic granules.

Pai agar, an egg-based medium, may be used instead of Loeffler’s.

Elek test principle

Tests C. diphtheriae for toxin production.

Principle: Immunodiffusion test. Line of precipitate forms in test agar when diphtheria toxin interacts with diphtheria antitoxin.

Procedure:

• Saturate strip of filter paper with diphtheria antitoxin

• Embed in special agar medium.

• Test MO, (+) & (=) control strains streaked onto agar.

• Streaks parallel to each other & perpendicular to filter
  paper.

• Incubate overnight at 35 C.

Interpretation:

• “Arc of identity”indicates test MO is toxigenic.

• No “arc” if nontoxigenic

Crynebacterium other than C. diptheriae

Is a NF of human, mostly is a contaminant. But can cause serious infections.

Presumptively ID with:

Catalase +

GS: small GP, pleomorphic rods.

Colony: small, white, grey or yellow.

Hemolysis: usually non-hemolytic.

What are the other 2 spp. of Corynebacterium that are watched?

C. jeikeium: Formerly known as CDC Group JK.

• Serious infections in immunocompromised patients.

• Presence of catheters/prosthetic devices contribute to
  infection.

• Causes septicemia and endocarditis – most common cause of diphtheroid prosthetic endocarditis in adults.

• Resistant to many antibiotics.

C. urealyticum:

• Resistant to antibiotics.

• Associated with urinary tract infections.

• MO hydrolyzes urea & cause stones in urinary tract.

Listeria monocytogenes

GS: small gpr, no spores. might even look coccobaccillary or coccoid.

Culture: faculative anaerobic, will grow on routine media.

NF: environment, vagina, intestines of healthy humans.

Diseases: in pregnant women to the baby from meats and dairy. Also immunocompromised and elderly.

Specimens: CSF, blood, vaginal secretions.

Hemolysis: Beta, remove colony to see.

Catalase +

Bile esculin: Positive.

Motility: motile at RT (25) (Umbrella) and reduced motility at 35 (tumbling appearance).

This can be confused with GBS and Corynebacterium.

Erysipelothrix rhusiopathiae

GS: GP, pleomorphic, no spores.

Culture: Faculative anaerobic, grows on routine media.

Found: soil, water, animals (swine).

Disease: Mostly animal pathogen and animal contact. Forms Erysipeloid (red skin lesion). May disseminate.

Specimens: skin and infected sites.

Catalase =

Hemolysis: Alpha, gamma

Motility: non-motile.

H2S production on TSI agar: +

Lactobacillus

GS: GPR, no spores, long rods in chains.

Culture: BAP, CHOC. atmosphere varies with strains.

Hemolysis: Alpha, gamma (just like listeria).

NF: oral cavity, GI, Female genital tract.

Diseases: rare.

Catalase =

Motility: non-motile

Use anaerobic media and procedures for full ID.

Arcanobacterium haemolyticum

Diseases: may cause pharyngitis, wound infections, bacteremia.

GS: GPR

Hemolysis: Beta.

Catalse =

Complete ID with Biochemical testing.