BACTE 1

PART 1

Phenotypic Characteristics

To Identify Bacterial Species:

• Observable Characteristics also known as ?

Microscopic Morphology

Colony Morphology

Antibiotic Resistance Pattern

Biochemical Test Results

Example of Observable Characteristics:

Microscopic morphology

What Observable Characteristic is this?

• We use staining microscopy

Colony morphology

What Observable Characteristic is this?

• We use Culture/Isolate

AST and Biochemical Test

We perform this to help Identify the species of bacteria

Biochemical Test

• can do it manually

• can do it automatically

• or semi-automated

VITEK system

What Biochem Test:

• automated viral identification

• will automatically give you the exact species

API (Analytical Profile Index Test)

What Biochem Test:

• kit

• has the microtubes that already have the substrate

• you only need to inoculate

• we input results sa computer kasi may program na mag identify for you

CSF

Example of Specimens accepted in the laboratory:

• Receive → CENTRIFUGE → Sedimentation → Gram Stain

• !TO RULE OUT MENINGITIS!

• Collected via Lumbar Tap (invasive procedure)

S. pneumoniae

H. influenzae

N. meningitidis

CSF Bacterias that can cause Meningitis:

(SpHiNm)

1. Spx Collected via invasive procedure (STAT)

2. Unpreserved

3. Quantitation (counting)

4. Preserved

If the laboratory is unstaffed what should be the flow of sample processing:

Urine

Example of Specimens accepted in the laboratory:

• !RULE OUT REPEATED UTI!

• Spx of Choice: Clean Catch Mid Stream Catheterized

• Anaerobic: Suprapubic Urine

• Inoculate Quantitatively

  • Use of CALIBRATED LOOP (not wood) = 1 / 1000 ul

  • 1 ul = DF: 1000 / 10 ul = DF: 100 ul

E. coli

K. pneumoniae

PPM

Urine Bacterias that shows Gram (-) rods [Enterobacterates]

(EcKpPPM)

S. saprophyticus

Enterococci

Urine Bacterias that shows Gram (+) cocci

• PEA Agar

• CNA Agar

(SsE)

Stool

Example of Specimens accepted in the laboratory:

• To !DETECT GIT PATHOGENS!

• “ISOLATE STREAKING“ = inoculate in 4 quadrants

• Non - Sterile Flora

Salmonella

Shigella

Stool Bacterias that can cause GIT:

(SS)

Sputum

Example of Specimens accepted in the laboratory:

• NON STERILE SPX

• How to check if its really sputum:

  • Bartlett’s Criteria (>25 mins, <10 secs)

  • Observe for Alveolar Macrophage Columnar Cells

• !DIGESTION & DECONTAMINATION!

Digestion

Liquify the sample to free any trapped organisms

Decontamination

To remove normal flora / contaminants

1. NaOH (2,3,4%)]

2. NALC + NaOH

3. Z-TSP

4. Oxalic acid

Reagents used in Digestion & Decontamination for Sputum:

NALC + NaOH

Gold Standard Reagent used in Digestion & Decontamination for Sputum:

1st one is the digestant , 2nd one is the decontaminant

Oxalyic acid

In sputum, helps in specimens likely to contain P. aeruginosa

M. tuberculosis

Sputum Bacteria:

• Book: 1 spx/day in 3 days [International Exams]

• DOH: 2 spx in 1 day [local boards]

  • 1st spx = early morning

  • 2nd spx = random

• “Mycobacterium” = “Slow Growers”

• Additional:

  • If may tumubo agad sa media hindi yan Mycobacterium, possible Normal Flora & Contaminants

Gene Xpert

USED IN SPUTUM SPX:

• Rapid and Sensitive

• Mostly additional test req. ng doctor

  • X ray

  • AFB smear

  • Skintest (Mantou x)

Blood

• Sterile, to !RULE OUT BACTEREMIA & SEPTICEMIA!

1. COLLECTION SITE

   • Cleans site w/ alcohol → Iodine Scrub → Alcohol rinse

2. PREPARE 2-3 SETS

   • One for aerobic, One for anaerobic

   • Collect blood Twice from both arms (1 hr interval)
     adults = 20 ml/arm

3. ANTICOAGULANT

   • Bacterial Culture: SPS

   • Viral Culture: Heparin

   • PCR (Blood): White top tube w/ EDTA

   • Not to be used in Micro = EDTA & CITRATE

S. epidermidis

FOR BLOOD:

Ex: Blood Culture Contaminants

• Aerobic = ?

(Se)

Cutibacterium acnes

FOR BLOOD:

Ex: Blood Culture Contaminants

• Anaerobic = ?

(Ca)

Direct Smear

Indirect Smear

A. STAINING & MICROSCOPY

• Examples of Smear Preparation

Direct Smear

UNDER SMEAR PREPARATION:

• from primary clinical specimen

• Directly from the specimen

Indirect Smear

UNDER SMEAR PREPARATION:

• growth from solid, semi-solid or broth media

Heat Fixation

• From the book: For Anaerobes we use Methanol for _______

Simple Staining

UNDER STAINING TECHNIQUES:

• Use of only 1 dye, the color of the dye is the resulting color

  • i.e. methylene blue

Negative Staining / Indirect / relief

UNDER STAINING TECHNIQUES:

• Only the background and not the organism is stained

  • i.e. india ink, Organism appears colorless

Special Staining

UNDER STAINING TECHNIQUES:

• to demonstrate special features of the cell

Hiss, Anthony’s, Tyler, Muir

UNDER SPECIAL STAINING:

• Stains Capsular stains

Dorner’s, Schaeffer and Fulton, Wirtz and Conklin

UNDER SPECIAL STAINING:

• Stains Spore stain

Schaeffer and Fulton

WHAT STAIN?

Primary Dye: Malachite Green

Counter stain: Safranin

Spores: Green

Other Structures: Red

Grays, Fisher and Conn, Leifson

UNDER SPECIAL STAINING:

• Stains Flagella

Albert’s, Neisser, Ljubinsky, Ponder,methylene Blue, Lindergran, Burke’s technique

UNDER SPECIAL STAINING:

• Stains Metachromatic granules

  • Babes Ernst Bodies = C. diptheriae

  • Much Granules = M. tuberculosis

  • Intracellular org like, Halberstaedter prowazek → glycogen containing → iodine = C. trachomatis

Wayson stain, Methylene Blue

UNDER SPECIAL STAINING:

• Stains Polar Bodies

Levaditi’s

UNDER SPECIAL STAINING:

• Stains spirochetes

Differential Staining

UNDER STAINING TECHNIQUES:

• to differentiate 1 organism from another

  • i.e. Gram staining and AF staining

Gram Staining

Acid Fast Stain

UNDER DIFFERENTIAL STAINING:

• Two examples of Differential staining

Reagent: Crystal Violet

Gram (+): Violet

Gram (-): Violet

Under Gram Stain:

• Purpose: Primary Stain / Initial Stain

• Reagent: ?

• Gram (+): ?

• Gram (-): ?

Reagent: Grams Iodine (pH: alkaline, fix the organism to stain)

Gram (+): Violet

Gram (-): Violet

Under Gram Stain:

• Purpose: Mordant

• Reagent: ?

• Gram (+): ?

• Gram (-): ?

Gram (+): Violet

Gram (-): Colorless

Under Gram Stain:

• Purpose: Decolorizer

• Reagent: Alcohol (ethanol) Acetone Acetone-alcohol mixture

• Gram (+): ?

• Gram (-): ?

Reagent: Safranin

Gram (+): Violet

Gram (-): Red

Under Gram Stain:

• Purpose: Counterstain Secondary stain

• Reagent: ?

• Gram (+): ?

• Gram (-): ?

Violet / Purple

Gram positive bacteria is stained ?

Red

Gram negative bacteria is stained ?

Decolorization

Most Critical step in gram staining ___________?

Fungi → Gram (+)

Hucker’s modification ____________________

• Add ammonium oxalate to crystal violet

0.1% Fuchsin

• __________ may be used in place of safranin to improve staining of some gram-negative organisms that are poorly stained with safranin like Bordetella, Legionella.

• In smear preparation air dry smear or use slide warmer set at 60 degC

• Do heat fixation or methanol fixation

  • ff: smear preparation

Neisseria, Moraxrella and Veilonella

RULES:

• All COCCI are Gram (+) except ? , ? , ?

(NMV)

Mycobacterium, Corynebacterium, Clostridium, Bacillus, Erysipelothrix, Lactobacillus, Listeria

RULES:

• All BACILLI are Gram (-) except ? , ? , ? , ? , ? , ? , ?

(MCCBELL)

Reported as Gram Negative

RULES:

• All SPIRAL ORGANISMS are reported as Gram ?

• Rickettsia; Chlamydia

• Mycoplasma; Ureaplasma

• spirochetes

RULES:

• Not GRAM stained are

1. _________, ___________ (intracellular);

2. _________, __________ (wall less);

3. __________ (can’t resolved by bright field)

False Gram (+)

False Gram (+) or Gram (-) ?

• Underdecolorization, use of thick smears

False Gram (-)

False Gram (+) or Gram (-) ?

• over decolorization, use of dry or old colonies, use of acidic mordant, mordant was omitted in the procedure

Mycobacterium

Acid Fast Stain is used for this bacteria ?

• Gordonia

• Rhodococcus

• Nocardia

• Teukamurella

Partially Acid fast Stain is used for these bacteria?

(GRNT)

Ziehl Neelsen / Hot method

• Method BEST FOR DSSM (direct sputum smear microscopy)

• under OIO

Kinyoun’s / Cold method

• Method best for DETECTING AFB ON TX

Auramine-Rhodamine (Fluorochrome)

• Method that is MOST SENSITIVE

Mordant

• Para tanggalin ang Mycolic Acid (temporary)

• Purpose in Acid Fast Stain

• CARBOL FUCHSIN

• AURAMINE-RHODAMINE DYE

UNDER ACID FAST STAIN:

• Purpose: PRIMARY

• Ziehl Neelsen / Hot method & Kinyoun’s / Cold method: ?

• Auramine-Rhodamine (Fluorochrome): ?

• HEAT / STEAM

• WETTING AGENT (TERGITOL)

• X

UNDER ACID FAST STAIN:

• Purpose: MORDANT

• Ziehl Neelsen / Hot method: ?

• Kinyoun’s / Cold method: ?

• Auramine-Rhodamine (Fluorochrome): ?

• 3% ACID ALCOHOL (HCL + EtOH)

• 0.5% ACID ALCOHOL

UNDER ACID FAST STAIN:

• Purpose: DECOLORIZER

• Ziehl Neelsen / Hot method & Kinyoun’s / Cold method: ?

• Auramine-Rhodamine (Fluorochrome): ?

• usually METHYLENE BLUE

• 0.5% kMnO4

UNDER ACID FAST STAIN:

• Purpose: COUNTERSTAIN

• Ziehl Neelsen / Hot method & Kinyoun’s / Cold method: ?

• Auramine-Rhodamine (Fluorochrome): ?

• Acid Fast = Red Against Blue Background

• Non-Acid Fast = Blue / Green (depends on the stain)

• Yellow Against Black bg

UNDER ACID FAST STAIN:

• Purpose: RESULT

• Ziehl Neelsen / Hot method & Kinyoun’s / Cold method:

  • Acid Fast = ?

  • Non-Acid Fast = ?

• Auramine-Rhodamine (Fluorochrome): ?

Malachite Green

May be used as substitute for Methylene Blue Counterstain:

• advantage:

  • Easier to read because of better contrast

  • will allow examination of smear under LPO

Mycolic Acid

✓ Acid Fastness is due to Hydroxymethoxy acid or __________

✓ Fix smear via heat fixaton, no heating for partially acid fast

Mycobacterium , Nocardia

RULE:

• All bacteria are Non-Acid Fast except: __________, slightly acid fast is _________

2×3 cm

Size of sputum smear ________ (AFB smear)

300 fields

Before reporting a truly negative result, examine at least _____ fields

Acridine Orange (sub for KMNO4)

Can be used as counterstain in Fluorochrome method __________

• Fluorochrome dye

• To stain cell wall-less bacteria (ex. ureaplasma)

• Mycoplasma (no cell wall) aka mollicutes = !smallest living organisms! , requires sterol for growth (cholesterol)

blue ; red

Other Methods of Acid-Fast Staining:

Pappenheim’s (urine) to differentiate M. smegmatis _______ from M. tuberculosis ___________

red ; blue

Other Methods of Acid-Fast Staining:

Baumgartens (tissue) to differentiate M. leprae __________ from M. tuberculosis _____________

Hematoxylin

Other Methods of Acid-Fast Staining:

Fite Faraco’s M. leprae – use of _____________ as counterstain

ave 0.4-2.0 u

Microscopically bacteria:

• size (ave ?-?)

• shape

• arrangement

• staining rxn

• motility

Listeria monocytogenes

Motility:

• True (presence of flagella)

• Brownian Motility

  • Pseudo motility exhibited by Normal Microorganism (random movement)

• To detect = Flagellar stains

  • use Hanging drop to identify ________

• Use semisolid media

Culture Media

Any medium that contains all what is needed to support bacterial growth

• 0%

• 0.5-1%

• 2-3%

• x

TYPES OF MEDIA AS TO CONSISTENCY:

Solidifying agent / agar:

• Liquid: ?

• Semi - solid (tube - butt): ?

• Solid (petri dish): ?

• Biphasic: ?

Liquid Media

TYPES OF MEDIA AS TO CONSISTENCY:

• These are example of ?

  • TSB - Trypticase Soy Broth

  • APW - Alkaline Peptone Water

  • BHI - Brain heart Infusion

Semi-solid medium

TYPES OF MEDIA AS TO CONSISTENCY:

• These are example of ?

  • SIM

Solid Medium

TYPES OF MEDIA AS TO CONSISTENCY:

• These are example of ?

  • Liquefiable: EMB, MSA

  • Non-liquefiable: Not agar based Rice Medium

Biphasic

TYPES OF MEDIA AS TO CONSISTENCY:

• These are example of ?

  • HBT - Human Blood Bilayer Tween

  • Castaneda’s

40-50C ; 80-90C

AGAR usually derived from red algae, solidifies at _______ & melts at _________

55-60C

Cooling temp dispensing of Agar ________

General Purpose Nutritive Media

TYPES OF MEDIA USED:

• Contains only what is needed to support bacterial growth

• For routine cultivation examples:

  • Nutrient Agar

  • SDA

Enrichment Media

TYPES OF MEDIA USED:

• To enhance bacterial growth, to increase bacteria yield

  • Selenite broth

  • Tetrathionate broth

  • APW-alkaline Peptone Water

Salmonella

UNDER ENRICHMENT MEDIA:

you can use this media for detection of ?

• Selenite broth

• Tetrathionate broth

vibrio

UNDER ENRICHMENT MEDIA:

you can use this media for detection of ?

• APW-alkaline peptone water

Enriched media

TYPES OF MEDIA USED:

• Contains Blood

• BAP, CAP

• For growing fastidious organisms like Neisseria, Haemophilus

Sheeps Blood

UNDER ENRICHED MEDIA:

• In preparation of BAP

  • Most Preferred ?

Human

UNDER ENRICHED MEDIA:

• In preparation of BAP

  • Least Preferred ?

Streptococci

UNDER ENRICHED MEDIA:

• In preparation of BAP

  • Sheep’s Blood used for ?

Haemophilus

UNDER ENRICHED MEDIA:

• In preparation of BAP

  • Horse Blood used for ?

G. vaginalis

UNDER ENRICHED MEDIA:

• In preparation of BAP

  • Human Blood used for ?

Trypticase Soy Broth

UNDER ENRICHED MEDIA:

• In preparation of BAP

  • For subculturing various bacteria

Selective media

TYPES OF MEDIA USED:

• Used to promote growth of a particular organism & at the same time preventing growth of other

• It contains inhibitor = Antibiotic, Dye (Crystal Violet)