Micro Week 1 Summary

List some normally sterile body sites

-Kidneys/ bladder

-CSF, blood, and other body fluids

-Deep body tissues or organs

-Eye

-Inner/ middle ear

-Bones and joints

-Lower respiratory tract

List some normally contaminated body sites

-Throat, mouth, upper respiratory tract

-Skin/ superficial wound

-Urogenital tract of females

-Lower GI tract

Describe 3 basic microscopic shapes of bacteria

-Bacilli: rods

-Cocci: spheres

-Coccobacilli: coccoid rods

-Spirals: curved rods

What term describes bacteria with a variety of shapes?

Pleomorphic

List 3 bacterial structures that do not stain by Gram stain

Capsules

Flagellae

Spores

Describe how the structures not stained by Gram stains may be detected.

-Capsules: Special capsular stain

-Flagellae: Stain, wet prep, or motility media

-Spores: Special spore stain

Based on their oxygen requirements, describe growth in THIO of aerobes, anaerobes, capnophile, and facultatives.

-Aerobes: Near the top

-Anaerobes: Near the bottom

-Facultatives: Throughout

-Capnophile: top 1/3

List, in order of use, the reagents used in a Gram stain.

-Crystal Violet

-Iodine

-Decolorizer

-Safranin

What is the purpose of Crystal Violet in a Gram stain?

Stains the organism

What is the purpose of Iodine in a Gram stain?

Acts as a mordant in Gram stains

Adhering the violet to the cell walls

What is the purpose of Decolorizer in a Gram stain?

Washes away primary stain from GNB cell walls

What is the purpose of Safranin in a Gram stain?

Counterstain

Explain why gram positive organisms stain purple and why gram negative organisms stain red/pink.

The thick peptidoglycan layer of gram positives retains the crystal violet staining purple, while the thinner cell wall of gram negs releases the crystal violet and is stained with the counterstain safranin which appears pink to red.

What is effect on the appearance of organisms on gram stained smears when: the decolorizer is left on too long

May cause a gram positive organism to show as gram negative

What is effect on the appearance of organisms on gram stained smears when: the decolorizer is not left on long enough

May cause a gram negative organism to show as gram positive

What is effect on the appearance of organisms on gram stained smears when: safranin/ carbol-fuchsin is left on too long (longer than 1 minute)

Will cause a gram positive organism to stain red/gram negative

What is effect on the appearance of organisms on gram stained smears when: the decolorizer is applied unevenly

The same organism will appear both gram positive and gram negative on the same slide

What is effect on the appearance of organisms on gram stained smears when: the smear is too thick with organisms

Can't properly decolorize

Gram variable result

What is the purpose of streaking PIM for isolation?

To isolate individual colonies

Especially in samples where there may be multiple organisms

Distinguish between the effect on bacteria of disinfectants and sterilization.

Disinfection - Kills all vegetative bodies Sterilization - Kills all vegetative bodies and spores

Describe the correct autoclave conditions for sterilizing most lab media or supplies

121 degrees C

15lbs

15 mins

Describe the temperature/time for using a drying oven for sterilization

160-180 degrees F

1-2 hours

What percent CO2 is produced in a CO2 pak/bag?

5-10%

Describe the action on SBA (Sheep's Blood Agar) of bacteria that are: Beta Hemolytic

Complete lysis of RBCs

Total clearing around the colony

Describe the action on SBA (Sheep's Blood Agar) of bacteria that are: Alpha Hemolytic

Partial lysis of RBCs

Greening on plate around the colony

Describe the action on SBA (Sheep's Blood Agar) of bacteria that are: Gamma Hemolytic

No lysis of RBCs on plate

Describe the gram stain appearance of normal flora organisms: Corynebacterium (Diphtheroids)

Gram Positive Bacilli: pleomorphic palisades, club-shaped and chinese letter forms

Describe the gram stain appearance of normal flora organisms: yeast

-Gram positive

-Round to oval

-Budding can have psuedohyphae

Give the purpose of the following media, and the main ingredients and their functions: SBA

Sheep Blood Agar

-isolate routine and fastidious bacteria

-observe hemolysis

-T-soy agar base

-5% sheep blood

Give the purpose of the following media, and the main ingredients and their functions: CNA

Colistin Naladixic Acid

-Selects gram positives

-inhibits gram negatives

-usually used for aerobic culture

-Columbia agar with sheep blood and antibiotics

Give the purpose of the following media, and the main ingredients and their functions: Chocolate (CA)

-peptone agar base

-2% hemoglobin or Isovitalex

-Incubated in CO2

-recovers more fastidious bacteria

-ex. Neisseria and Haemophilus

Give the purpose of the following media, and the main ingredients and their functions: EMB

Eosin Methylene Blue

-peptone agar base

-lactose (dark purple if + and clear if -)

-selects for gram negatives

-inhibits gram positives

-differentiates GN based on lactose utilization

-Levine's also has sucrose and may show green sheen

Give the purpose of the following media, and the main ingredients and their functions: MAC

MacConkey

-peptone agar base

-lactose (red/pink = + and clear = -)

-neutral red indicator

-crystal violet and bile salts inhibitors

-selects for gram negatives (enterics)

-inhibits gram positive

-differentiates GN based on lactose fermentation

Give the purpose of the following media, and the main ingredients and their functions: MSA

Mannitol Salt Agar

???

Differential media

Give the purpose of the following media, and the main ingredients and their functions: MTM

Modified Thayer Martin

-enriched CA

-antibiotics: colistin, nystatin, vancomycin, trimethoprim

-selects for Neisseria gonorrhoeae and Neisseria meningitidis

-inhibits normal flora

All members of Micrococcaceae/Staphylococcaceae are gram ______________ __________in ______ ,_______ or ___________. All are Catalase ______.

Gram Positive Cocci

Pairs, Clusters, Tetrads

Positive

Staph vs Micrococcus: glucose, bacitracin, microdase

-Staph: Glucose Fermenter, Bacitracin Resistant, Microdase Negative

-Micrococcus: Glucose oxidizer, Bacitracin sensitive, microdase positive

Describe Staph aureus and Micrococcus colonies, giving two major ways they differ

-Staph: Gold/Tan often Beta hemolytic

-Micro: Bright Yellow

Give the gram stain appearance of Staphylococcus

Gram Positive Cocci in pairs, clusters, or tetrads

Give the catalase test result for Staphylococcus

Bubbling; Positive

What is the catalase test reagent?

peroxidase

What may cause a false positive catalase test in lab?

Picking up some of the agar

What biochemical test best distinguishes Staph aureus from the other normal flora staph?

Coagulase

When is the slide vs tube coagulase test done?

-Slide coagulase is done to detect bound coagulase faster

-Tube coagulase is done to detect free coagulase more accurately

What causes a false negative coagulase test, and why?

-Over shaking or agitation (clot shrinks)

-Over incubation

What rapid test often done in labs replaces the coagulase test?

Latex Agglutination test that detects bound clumping factor

Describe the color/reaction on MSA of: Staph aureus

Mannitol +

(Yellow)

Describe the color/reaction on MSA of: Staph epidermis

Mannitol -

(Red)

Describe the color/reaction on MSA of: Staph saphrophyticus

Mannitol +/-

Give the expected results for Novobiocin testing for: Staph saprophyticus

Resistant

Give the expected results for Novobiocin testing for: Staph epidermidis

Sensitive

If a staph species is beta lactamase positive, then what antibiotics should not be used to treat its infections?

Penicillins

Cephalosporins

Review how the rapid beta-lactamase test works

Rapidly detects the presence of the beta-lactamase enzyme

Describe MRSA/ORSA strains

Strains resistant to synthetic penicillins

What is the disk currently used to detect MRSA/ORSA?

Oxacillin

Describe a Chromagar screen for MRSA/ORSA and VRSA screens.

• Purpose: Screen for MRSA carriers

• Specimen: Anterior nares/other

• Media: contains Cefoxitin, other inhibitors and a chromagenic substrate

• Principle: MRSA will produce an enzyme that hydrolyzes the chromagenic substrate, resulting in mauve-colored colonies or denim blue colonies—depends on media/company used

List the 5 most common general infections caused by Staph aureus and give the best specimen

-Scalded Skin Syndrome: wound

-UTI: urine

-Systemic TSS: blood, CSF

-Nosocomial Pneumonia: sputum

-Osteomyelitis: bone

Which enzyme is the basis for the coagulase test and aids in resisting phagocytosis?

coagulase

Which enzyme causes a false positive coag test?

Staphylokinase?

Which enzyme causes the clearing of methyl-green media?

?

Which enzyme is known as the spreading factor?

hyaluronidase

Which toxin causes scalded skin syndrome?

exfoliatin

Which toxin causes staph food poisoning?

enterotoxin

Which toxin causes toxic shock?

TSST

Describe the types of infections and populations most often associated with coagulase negative staphylococci: Staph epidermidis

UTI (cath)

IV cath

Shunts

Total joints

Heart valve surgery

Bacterimia

Endocarditis

Describe the types of infections and populations most often associated with coagulase negative staphylococci: Staph saphrophyticus

UTI (in sexually active young women and old men [catheter])

The scientific name for Group A strep is ____________

Streptococcus pyogenes

Describe typical colonies/hemolysis of Group A strep:

Tiny/ Pinpoint

Translucent

Wide beta hemolysis

Based on Group A infections, give two patient specimens most commonly plated

Throat

Wound

List/give results of two biochemicals tests used to identify Group A strep colonies

PYR: positive

Bacitracin: Sensitive

Describe the serological test often used to rapidly ID Group A strep culture isolates

Method: Latex Agglutination

Antigen: Group A polysaccharide

Antibody: Anti-Group A polysaccharide

List two sequellae infections of Group A strep

Rheumatic fever

Glomerulonephritis ASO and Streptozyme; test for virulence factor

ID the serological test used to detect Group A strep

Streptozyme

Antigen: Streptolysin O

Antibody: Anti-Streptolysin O

List 2 additional strongly beta-hemolytic strep that may be isolated from throat/wound specimens

Group C strep

Group F strep

Group G strep

How can Group C, F, and G strep be identified serologically?

Grouping of polysaccharide walls (Lancefield)

Give the SXT test/result that can distinguish Group C, F, and G from Group A strep

Group A and B strep are SXT resistant whereas other hemolytic strep are susceptible to SXT

Name the specimen that can be used for the Direct Antigen Detection of Group A strep directly from the patient.

Throat swab

Describe the colonies/hemolysis of Group B strep

Small

Grey/ White colonies

Narrow zone of hemolysis

Based on infectious caused by Group B strep: list 2 neonatal specimens that might contain them

CSF/ Blood

Urine

Based on infectious caused by Group B strep: list 3 maternal specimens that might contain them

Vaginal

Rectal

Cervical

Give two common species of Group D Enterococcus

Enterococcus facaelis

Enterococcus faecium

Give the most common species of Group D Streptococcus

Streptococcus bovis

Describe the colonies/hemolysis of typical Group D streptococci.

Medium

Greyish

Alpha or Beta hemolysis

Based on the infections caused by Enterococcus, list 3 common patient specimens for isolating it.

Wounds from bellow the waist

Blood/ Heart valve

Urine

List the most common specimen to isolate Streptococcus bovis

Blood/ Valve

List/give 3 test results for differentiating Group D streps/other strep: Enterococcus spp. Streptococcus bovis

-Entero:

6.5% Salt: Tolerant

PYR: +

Bile Esculin: ?

-Strep:

6.5% Salt: Intolerant

PYR: -

Bile Esculin: ?

Which of these tests is most commonly performed in the clinical lab?

PYR

Which strep are routinely resistant to Penicillin and other usual gram positive antibiotics?

Strep pneumo

Enterococcus

What does VRE stand for, and what populations are screened for VRE?

Vancomycin Resistant Enterococcus

Individuals housed in nursing homes/ facilities

Give the scientific name of the most commonly isolated alpha strep pathogen

Streptococcus pneumo

Distinguish between the colonies/hemolysis of: Strep pneumo and Strep viridans

Pneumo: Wide Alpha hemolysis

Small/ Large

Mucoid; pennies on a plate

Viridans: Alpha/ Gamma

Tiny

White/ Grey granular

List and give results of two biochemical tests that differentiate: Strep pneumoniae and Alpha strep

Pneumo: Optochin Sensitive

Bile Soluble

Alpha: Optochin Resistant

Bile Insoluble

Based on infections it causes, give 3 adult and children patient specimen most likely will yield Strep pneumo.

Sputum

CSF (elderly/ children)

Blood (urine/rapid)

Give 2 more children specimens that could yield Strep pneumo

Ear (Upper respiratory)

CSF

Give the biochemical used in the Taxo P/Optochin test.

Ethylhydrocupreine hydrochloride

What is the basis for the serological ID of Strep pneumo culture isolates?

Latex Agglutination

Antigen: Pneumo capsule

Antibody: Anti-Pneumo capsule

NOTE* Direct testing urine EIA are very common now as patients with Pneumonia shed antigen in the urine

Describe the modified Kirby-Bauer/Etest anti-susceptibility testing routinely done on Strep pneumo isolates?

Pneumococcus: test for penicillin

resistance: (1)Etest or

(2)1.0ug oxacillin disk-SBA in CO2/350C

(3) MICs (i.e., automated systems)

Describe a sputum smear from a patient with pneumoniae caused by Streptococcus pneumoniae (include cells & bacteria)

Squamos Epis

PMNs

Gram Positive diplococci (safety pin appearance)

Name two infections most likely caused by alpha strep (viridans) pathogens, and the most common specimen for detecting each

Dental Caries: Mouth/ Sputum

Endocarditis: Blood/ Valve

Satelliting strep grow on SBA only if the are satelliting colonies of _________________or if a ______________________ disk is applied to SBA

Staph aureus

Pyridoxal