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1. MICROBIOLOGY AND PARASITOLOGY

Created @November 12, 2022 8:13 AM

Last Edited Time @December 18, 2022 6:56 PM

Type SEMINAR LECTURE AND NOTES

Created By @louiden

Participants

Goals
TAKE DOWN IMPORTANT DETAILS IN MICRO

MEMORIZE IT BY HEART

Action Items
BACTERIOLOGY

MYCOLOGY

VIROLOGY

PART 1

BACTERIA SAID TO BE PROKARYOTIC -NO TRUE NUCLEUS

Cell wall less: Mycoplasma and Ureaplasma
Some produce spores (the best way to destroy spores is through autoclaving)
Genus: Bacillus and Clostridium “SPORE FORMING ORGANISM”
The average size of bacteria: 0.4-2.0UM
Considered as the smallest (Mycoplasma)

Considered the largest pathogenic organism (Bacillus anthracis)

Why bacteria is capable of causing disease because of: VIRULENCE FACTOR

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 TOXINS
EXOTOSINS: PRODUCE BY GRAM-POSITIVE EXCEPT Listeria spp, and maybe also produced by gram-
negative.
Protein in nature, Unstable in heating
Once it is released in the body: the effect is local
Excreted by living bacteria

Ex:

TSST-1: S. aureus
Diphtheria toxin = C. diphtheria
Botulinum toxin (neuro toxin) = C. botulinum
Coagulase

ENDOTOXINS: Produce by Gram-negative organism

Release only by cell lysis ( cell death ): The effect is systemic or generalized
there are lipopolysaccharide in nature
Ex: Shigella

PART 2
CELL WALL
is also known as the Murein layer/Peptidoglycan(main component) layer

The usual site for the antibiotic action

Disaccharides: N-acetyl-glucosamine & N-acetyl muramic acid

Basis of GRAM STAINING ( Do not perform GS on Ureaplasma and Mycoplasma )

Gram-Positive Cell Wall: Teichoic acid with Thicker peptidoglycan

Gram Negative: no Teichoic acid with thinner peptidoglycan

CAPSULE

Prevent phagocytosis (antiphagocytic)

Considered a virulence factor

Contributes to the mucoid characteristics of colonies

TEST FOR CAPSULAR SWELLING TEST: NEUFELD QUELLING

Capsule components

w/ Polysaccharide Capsule: S. pneumonia, KPN, and N. meningitidis

w/ Polypeptide D-glutamic acid: Bacillus anthracis

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 w/ Hyaluronic acid: Pasteurella multocida

w/ Polyribosyl Ribitol phosphate: H. Influenza

w/ Alginate capsule: P. aueroginosa

Alginate is the virulence factor of PAE

Glycocalyx - Less organized compared with capsule, a slimy layer containing polysaccharides: S. mutans

Pili (Fimbriae)

Usually found on Gram Neg organism

COMMON PILI: for attachment/adhesion to host cell

SEX PILI: for gene conjugation, transfer genetic material

Conjugation is a mechanism for gene transfer

Can be a virulence factor

SPORES OR ENDOSPORES

Resistant structures that enable the organism in injurious conditions

An organism that is capable of producing spores: BACILLUS OR CLOSTRIDIUM

best way to eliminate: AUTOCLAVE

TERMINAL SWOLLEN SPORES: Clostridium tetani

CENTRALLY LOCATED SPORES: B: Anthracis

SUB-TERMINAL SPORES: C. Botulinum

💡 Board exam: the target of sterilization is SPORES

FLEGALLEA

Motile: Spiral and Bacilli

Organ for locomotion

for Spiral organisms, it is called AXIAL FILAMENTS OR PERIPLASMIC FLAGELLA.

How do we know if the organism is motile or not?

HANGING DROP TEST: Presumptive for Listeria monocytogenes

Flagellar Stains: Grays, Leifson

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 Semi-solid media: SIM (Sulfide Indole Motility & MIO (Motility Indole Ornithine)

Ex:
Motility is best observed at Room Tempt

Tumbling Motility: L. monocytogenes

Twitching Motility: Kingella kingae

Gliding/Sliding: Capnocytophaga gingivalis

Darting Motility: Campylobacter spp.

Shooting Star: Vibrio cholerae

Corkscrew Motility: Sphirochete s

INCLUSION BODIES

Represent store food

Metachromatic granules - Babes Ernst Bodies Volutin is seen in Corynebacterium diphtheriae

MUCH granules: Mycobacterium Tuberculosis

Halberstaedter prowazek: Glyocgen containing inclusion bodies seen in: Chlamydia trachomatis

Visualize using Jimenez: Macchiavelo and Castaneda

Cell membrane: NO STEROL except in MYCOPLASMA

SPECIMEN COLLECTION AND HANDLING

When to collect specimens? DURING ACUTE PHASE (within 72 hours of the disease)

Observe: Aseptic technique

Collect Adequate amount

The collected sample must be transported without delay 30 minutes to 2 hours.

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 💡 IMPORTANT TO MEMORIZE!!

Priorities the Body fluids!

BLOOD

Purpose of doing blood culture: roll-out bacteremia or sepsis

The mere presence of bacteria in the blood: Bacteremia

Sepsis there is a systemic inflammatory response.

Blood Pathogens: PAE, S. aureus, E. Coli (PSE)

Biomarker for fungemia: C. Albicans, Potential biomarker for Sepsis: Procalcitonin (PCT)

The phlebotomy site must be cleansed with: 70-95% alcohol → Iodine scrub → ALCOHOL RINSE

Common contaminants: P. acnes, S. epidermidis. Diptheroids. Viridans Strep

Dilution of blood to the medium: 1 is to 10 or 1:10

Anticoagulant: SPS 0.0025%

Disadvantage: Inhibits Neisseria. G Vaginalis. Diphtheroids, Streptobacillus moniliformis

(NGDIS)

Counteract: add with 1% gelatin

Not to be used for bacteriologic Culture: EDTA (can be used for viral culture), Heparin (inhibits gram-
positive or yeast),

Blood culture bottles: 7 Days

For brucellosis detection: is 3-4 weeks; for Leptospirosis - it’s 8 weeks

Endotracheal aspirate: specimen of choice when the Px is intubated

CSF (Critical or invasive)

Collected in 3 tubes, used tube #2 for micro

Storage tempt: 37 Degrees, Transport tempts: Room Temperature

For Smear preparation & culture:

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 Centrifuge

Remove supernatant

WE USD THE SEDIMENTS BOARD EXAM QUESTION

URINE:

Specimen of choice: Mid-Stream Clean Catch

#1 CAUSE OF UTI: E COLI

UTI IN YOUNG FEMALE: S. SAPHROPHYTICUS

Perform colony count using CALIBRATED LOOP to determine # of colonies /mL of urine

Considered UTI in colony count: s

Formula:

# of colonies counted x dilution factor = colony/mL of urine

Dilution factor

if 1 uL loop was used: x1000

if 10 uL loop was used: x100

SPUTUM

Evaluate quality through, Bartlett’s Classification: No number of Squamous epithelial and PMNS

If >10 SECs but <25 PMNs = Not suitable/Not acceptable.

For M. Tuberculosis identification.

Purpose of Decontamination: remove contaminates and removes normal flora.

Why? because M. tuberculosis is a slow grower

Purpose of Digestion: Is to liquefy the mucus or free any trapped organism

The gold standard for digestion and decontamination: N-acetyl L-cysteine and Sodium Hydroxide

Others: Z-STP

4% NaOH

Cetylpyridium chloride sodium chloride method.

5% oxalic acid: for specimen that contains pseudomonas and proteus

M. Tuberculosis is classified as a BSL III pathogen, Culture must be done using BSCL II

Considered agent for community-acquired pneumonia (CAP) is Klebsiella pnuemoniae

Considered as the most cause of ventillator-acquired pneumonia: P. aureginosa

THROAT SWAB

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 Identification of S. pyogenes major throath pathogen

NASOPHARYNGEAL SWAB

Normal throat flora: VIRIDANS STREP

Required to detect Neisseria meningitidis and also B. pertussis and H. Influenzae

Toxic to Neisseria: Cotton Swab

Toxic to viruses: Calcium alginate

STOOL

Detection of GIT Pathogens like:

Maybe collected if stool collection is not possible

Number of quadrants on plated media: 4

GS is not usually done.

One way to identify species is to check their morphology under microscope

Staining of Bacterial Cells

Direct Staining - uses only one dye, the color of the dye is the resulting color

Indirect Staining/Negative or Relief Staining - Only the background and not the organism is stained “INDIA
INK”

💡 Board exam Question: WHAT WILL BE THE COLOR OF THE ORGANISM IN INDIRECT
STAINING
ANSWER → COLORLESS

In Schaeffer and Fulton

Used as a primary dye: Malachite green
Counter stain: Carbol fuchsin
Spores will appear: Green

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 Differential Staining:

requirement: bacterial smear, dry and heat fixation

Gram Staining

GRAM
PURPOSE REAGENT GRAM NEG
POSITIVE

PRIMARY STAIN CRYSTAL VIOLET VIOLET VIOLET

MORDANT GRAMS IODINE VIOLET VIOLET

95% ALCOHOL, ACETONE OR ALCOHOL ACETONE

DECOLORIZER VIOLET COLORLESS
MIXTURE

COUNTER
SAFRANIN VIOLET/PURPLE RED
STAIN

CONTINUATION

MOST CRITICAL STEPS IN GRAM STAINING: DECOLORIZATION

HUCKER’S MODIFICATION: FOR FUNGI

AMMONIUM OXALATE + CRYSTAL VIOLET

CARBOL FUCHSIN CAN BE USED AS A COUNTERSTAIN AS A PLACE OF SFRANAN. USED TO I MPROVE

SOME GRAM-NEGATIVE ORGANISMS THAT ARE POORLY STAINED WITH SAFRANIN.

ACID-FAST STAINING

ZIEHL NEELSEN KINYOUNS COLD AURIAMINE BAUGMGARTEN’S

PURPOSE FITE FARACO
HOT METHOD METHOD RHODAMINE TAIN

CARBOL CARBOL AURAMINE-

PRIMARY
FUCHSIN FUCHSIN RHODAMINE DYE
WEETING WEETING
MORDANT AGENT - AGENT -
TERGITOL TERGITOL

3% ACID 3% ACID
ALCOHOL ALCOHOL
DECOLARIZIER COMBINATION COMBINATION 0.5% ACID ALCOHOL

OF HCL AND OF HCL AND

ETHANOL ETHANOL

METHYLENE METHYLENE 0.5% POTASSIUM

COUNTERSTAIN PERMANGANATE
HEMATOXYLIN
BLUE BLUE

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 ZIEHL NEELSEN KINYOUNS COLD AURIAMINE BAUGMGARTEN’S
PURPOSE FITE FARACO
HOT METHOD METHOD RHODAMINE TAIN

ACID-FAST:
ACID-FAST: RED
ACID-FAST: RED YELLOW
NON-ACID FAST: MTB: BLUE M.
RESULTS NON-ACID FAST: AGAINST BLACK
GREEN OR leprae: RED
GREEN OR BLUE BACKGROUND
BLUE
NON ACID:
SUBSITUTION
FOR MALACHITE MALACHITE
METHYLENE GREEN GREEN
BLUE

All bacteria are non-acid fast except for Mycobacterium

Weak acid fast: Nocardia spp.
Responsible for the acid-fast is: MYCOLIC ACID/HYDROXYMETHOXY ACID (lipid)
ACID-FAST STAINING

ZIEHL NEELSEN = DSSM

KNYOUNS METHOD = AF IN TISSUES

FLUOROCHROME METHOD = MOST SENSITIVE

Note: look for acid-fast organisms through OIO

if you use the fluorochrome method: LPO

Size of AFB smear: 2x3cm

TUBE MEDIA PLATED

WEIGH DISSOLVE DISPENSE STERILIZE WDDS WEIGH DISSOLVE STERILIZE DISPENSE WSDS

LIQUID SEMI-SOLID SOLID BIPHASIC

SOLIDIFYING
0 0.5-1% 2-3%
AGENT/AGAR

HBT (Human Blood bi-layer

TSB ALKALINE LIQUEFIABLE: EMB MSA
tween) for G. vaginalis
EXAMPLES PEPTONE SIM NON LIQUIFIABLE: RICE
Castaneda medium (brucella
WATER BHI MEDIUM (media for fungi)
spp).

General purpose: Contains only what is needed:

Ex:
Nutrient Broth, Nutrient Agar

Enrichment media: Enhance bacterial growth

Alkaline peptone water - vibrio spp
Selenite broth
Tetrathionate broth

Enriched Media: for the fastidious organism, contains blood

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 BAP: for Streptococci
CAP(heated blood agar): Neisseria and Haemophilus
Horse blood: for Haemophilus
Human blood: G. vagenalis

Selective Media: Promotes growth of particulate organisms, contains inhibitor. Prevent growth of
UNWATEND ORGANISMS

CTBA
Inhibitor: Potassium tellurite
For isolate of C. diphtheriae

MAC
Inhibitor: Crystal violet
For isolation of: Gram-negative

Differential media: a type of media that is used to differentiate organisms that are growing together

EMB
MAC
TCBS: differentiate if sucrose or nonsucrose fermenter

Selective and Differential:

EMB:
MAC:
TCBS: Selected media for vibrio spp.

3 general methods in identification methods

Manual: MTOD PREPARATION
Semi-automated:
API - Analytical profile index:
Uses plastic strips & microtubes with biochemical substrates.
The biochemical substrates are inoculated with pure culture suspension.

API 20E: Commercially available kit, specific for Enteric Pathogen.

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 API 20A: Specific for anaerobic pathogen

Automated: VTEK, MALDI-TOF

Automation program for bacteria identification and AST

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