BACTERIOLOGY I || Megan Arceño | Cloie Casinillo | Angela Magsucang A.Y.

2015 – 2016, 1st semester

EXERCISE NO. 1 - Much’s granules: Mycobacterium tuberculosis

- Bipolar bodies: Yersinia pestis (safety pin appearance)
Bacterial Morphology and Types  
Metachromatic granules on both poles

 Morphology  Flagella
- Size, shape, cell inclusion & gen. arrangement - Protein filaments; extend like long tails
- Used w/ other properties to identify bacteria - Component: flagellin
- Determined by mechanism of cell wall assembly - Motility: 25° C
- Altered by antibiotics (penicillin) - Mordant: tannic acid
- Has filament and hook
 Binary fission - Arrangement:
- Reproduction of bacteria   
Atrichous (non-motile; no flagellum)
 Types:  
Monotrichous (V. cholera; single flagellum)


  
Lophotrichous (Pseudomonas; tuft of flagella at one pole)
Shape Morphology Example Description 
 
Amphitrichous (L. monocytogenes; tumbling motility; flagella at both
Staphylococci S.aureus Grapelike clusters poles)
Streptococci S. hemolyticus In chains 
 Peritrichous (E. coli, S. typhi, P. vulgaris, P. mirabilis; rapid
Diplococci darting motility)
S. pneumonia - Parts:
Cocci -intracellular In pairs
N. gonorrheae   
-extracellular Tail
  
Sacrina S. luteus Packets of 8-more Body
 
Tetrads G. tetragena In fours Head

Sporingstreptobacilli B. subtilis ---
Non-sporing - H antigen (flagellar antigen)
Bacilli Snapping diplobacilli M. tuberculosis --- - Observed in: hanging drop method and semisolid solid
Slipping diplobacilli - Some undergo “phase variation”
 
Coccobacilli E. coli Short rod-shaped Ability to switch flagellar antigen

Curved-rod/
Vibrio V. cholerae
comma shaped  Slime layer
- Small/ thin layer of accumulated polysaccharide outside bacterial cell wall
Spiral T.pallidum
Spirochete Tighter waves - Composition: polysaccharide
T. vincentii
- Example: Sarcina lutea
Spirillum S. minus Coarser waves
- Protects bacteria from phagocytosis
- All bacilli have slime layer, except: B. anthracis
 
Layer: Poly-D-glutamic acid
 Genera of Spirochete: Treponema, Borella, Leptospira 
 Capsule
EXERCISE NO. 2 - Well defined structure of polysaccharide
- Composition: polysaccharide
Structures and Composition of Microorganisms - Protects from phagocytosis
- Slimy color in agar
 Granular inclusion bodies - Example: K. pneumoniae
- Function: storage vessel for nutrients
- Component: polyphosphate
- Intrachromatic granules/ polar bodies/ cytoplasmic bodies
- Babes Ernst granules: Corynebacterium diphtheriae
Page 1 of 21
 BACTERIOLOGY I || Megan Arceño | Cloie Casinillo | Angela Magsucang A.Y. 2015 – 2016, 1st semester

EXERCISE NO. 3  Intravital staining

- Motility is best observed
Examination of Bacteria in Fresh Preparation - Appears purple
- 1:2000 aqueous solution of crystal violet
 Unstained bacteria  Inhibits killing of bacteria
- Difficult to observe because of contrast between the cell and its  Diluted so as not to kill bacteria
surroundings
 Use wet mount to observe motility  Viewing preparation
- Hanging drop - LPO with diaphragm partly open
 Motility, binary fission, interrelationship of bacteria from one - Condenser slightly lowered
another - Switch to LPO very slowly to avoid breaking the cover slip

 Materials:  BEST GUIDE: side of drop

 Concave slide
o Normal slide dries up specimen
 Cover glass
o Where loop or bacterial suspension is placed PREPARATION OF BACTERIAL SMEAR
 Alcohol lamp
 Wire loop  Bacterial smear
 Vaseline - Dried preparation of bacteria cells on glass slide
o Prevents evaporation - Preparation:
1 Label glass slide on frosted edge (pencil only)
 Aseptic technique  Patient’s name
- Heat wire loop at 45° angle  Date
- To avoid cross contamination  Stain
- Steps: 2 Heat glass slide; hold along edges of glass slide to prevent

Sterilize inoculating loop by passing thru hottest part of flame contamination

Allow loop to cool (1-2 min) 3 Heat wire loop (dry heat sterilization)

Do not set loop down on any surface to avoid contamination 4 Flame mouth of culture tube
5 Shake bottle suspension and place 5 loopfuls
 True Motility: moves in one direction only 6 Make smear

- Proteus mirabilis; Proteus vulgaris ½ - 1 inch; CONCENTRIC CIRCLE GOING OUTWARDS
- Mechanism: and air dry
 Flagella
 Spirochete 7 Air dry
 Gliding motility  Preserve morphology
8 Heat fix
 Brownian Movement: Non-motile; erratic movement  Pass flame 3-5x

- Staphylococcus aureus; Gaffkya tetragena Smear side facing up

- has a colliding effect that makes it appear to jiggle because of: Reasons for heat fixing:
 surface tension o Remove excess water
 water pressure o Kill bacteria, but preserve morphology
o For better adherence
o For bacteria not to be washed off
o To soften cell wall

Page 2 of 21
BACTERIOLOGY I || Megan Arceño | Cloie Casinillo | Angela Magsucang A.Y. 2015 – 2016, 1st semester

 
Properly processed: o Give a sputum sample
o Evenly spread out on slide   
Spot-morning-spot sputum collection
o Not washed off of slide o Spot: initial visit to clinic
o Morphology not distorted o Morning: early morning before visit to clinic
- Smear on agar slant/ agar plate o Spot: during 2nd clinic visit; delivery of sputum sample
  
Appearance:

Small amountof bacterial growth is transferred to drop of H2O
on glass slide o Purulent portion
 
Use wire loop: 
o Prevents too large inoculum (common error)  of dust cells/ macrophage (>25
Presence
WBCs)
o Pace 1-2 loopfuls of sterile distilled H2O;  
Saliva portion (<10 SECs)
sterilize wire needle
EXERCISE NO. 4
 Acid Fast Bacilli smear
- Stained/ dried for observation purposes Differential Stains
- Helps in identifying morphology
- SMALL CONCENTRIC CIRCLES I. GRAM’S STAINING (Hucker’s Method)
- Smear size: 2cm (width) x 3cm (length) - Developed by: Hans Christian Gram
- Quality assurance: - Presumptive diagnosis with the specimen submitted
1 Screen area size - Reagents:
2 Evenness of smear (sputum fully & finely distributed)  Crystal violet/ Gentian violet (violet)
3 Thickness of smear - Primary stain: initially stain the smear
- Flood smear: 1 minute
 Bacterial suspension  Gram’s Iodine (betadine color)

1 Using sterile wire loop, touch 4-5 isolated colonies of the colony - Mordant: increase affinity of primary stain
morphology to cell wall of bacteria
2 Suspend to 2 ml Aline solution - Brownish color
3 Vortex to create smooth suspension - Flood smear: 1 minute
4 Compare with 0.5 McFarland Standard (tube #5) and adjust
  Acetone alcohol (colorless)
turbidity - Decolorizer: differentiator between gram
(+) and (-)
 Hints and Precautions - Most important step
- Inoculating loop must be cool before inserting to broth - Flood smear: 5 seconds
- Heat fix: smear is top side; always wait til dry  Safranin (red)
- Solid media: bacteria is sufficient 
- Counterstain: stains background and
bacteria not reactive to primary stain
 Tuberculosis: M. tuberculosis - Flood smear: 45 seconds
- Specimen collection - CVI (Crystal Violet Iodine) complex

 
Container specifications o
50 mL Gram (+) Gram (-)
o Translucent/ clear 1st Stain Violet Violet
 o Single use combustible mat Mordant Violet Violet
o Screw capped w/ water tight seal Decolorizer Violet Colorless
 o Easily labeled walls
  Counterstain Violet Red
Getting a sample specimen
Peptidoglycan later w/ teichoic acid Lipopolysaccharide
o Clear mouth
o Breath in-out 3x
Page 3 of 21
BACTERIOLOGY I || Megan Arceño | Cloie Casinillo | Angela Magsucang A.Y. 2015 – 2016, 1st semester

- Gram (+) bacteria tends to be gram (-) EXERCISE 5

1 Old specimen
The Isolation of Pure Culture of Organisms
2 Dyeing of specimen
3 Using acidic stain
4 Technical error
  Culture methods:
- Gram (-) bacteria tends to be gram (+) I.Pour plate
- Increased decolorization II. Streak plate
- Thick smear  Microbial culture

 Multiply a microbial organism by letting them reproduce in a
II. ACID-FAST STAINING (Ziehl-Neelsen Method)  culture medium under controlled lab conditions
- Principle: lipid-barrier   Primary diagnostic method
- Heat serves as mordant
  Bacterial colony
 Heat softens mycolic acid: stain enters  Visible cluster of bacteria growing on surface or within culture medium
- Reagents: 
  Culture medium
 Primary stain: Carbolfuchsin (red)
- Flood smear: 3-5 mins   Nutrient medium used for growing bacteria
 Mordant: heat  Types:
- Heat softens mycolic acid: stain enters
  
Broth/ liquid enrichment/ transport medium
 Decolorizer: Acid alcohol (colorless)   
 Semisolid 0.5 – 1% agar
 Loeffler’s methylene blue (blue)   
Solid 2-3% agar
- Stains background “blue”
- Also known as: Loeffler’s Alkaline Methylene Liquid Semisolid Solid
blue Observe colony
- Good quality smear depends on: Fermentation
Large # of microorganisms in appearance
 staining technique
fermentation studies Detect bacterial
 cleanliness of slide Pure culture isolations
motility
- Smear reading:
Promote
  Horizontal scanning Biochemical tests Storage of cultures
anaerobic growth
 Vertical scanning
Observe specific
biochemical reactions
III. COLD KINYOUN STAINING
- Uses M. tuberculosis bacteria
- Increasing phenol concentration  According to manner of dispensing:
- Wetting agent: tergitol  
 Plated culture medium: petri dish
- Reagents:  
Tube culture medium: test tube
  Primary stain: Kinyoun carbolfuchsin
- Carbolfuchsin, 95% alcohol, liquefied
phenol
 Mordant: heat Plated Tube

 Decolorizer: Acid alcohol Weigh Weigh

 Methylene blue Dissolve Dissolve
Sterilize Dispense
Dispense Sterilize

Page 4 of 21
 BACTERIOLOGY I || Megan Arceño | Cloie Casinillo | Angela Magsucang A.Y. 2015 – 2016, 1st semester

  Pure culture  Petri dish is inverted for incubation to prevent condensation/ moisture
  Single type of organism from falling onto surface of the plate which may interfere with
  Obtained artificially  bacterial colony formation
 GOAL: isolate a single specie of bacteria from a mixed population   Swirling: for even distribution
  QUEBEC COLONY COUNTER
I.Pour Plate Method  
 Device used to count the # of bacterial colonies
  Quantitative technique of counting bacteria
 REMINDERS
 USES: melted nutrient agar  
   Get 3 loopfuls and apply loopful to 1st quadrant only
 Single culture medium for isolation of non-fastidious organism  
   After flaming, always let the wire loop cool before streaking
 Composition  
 Touch 2nd and 3rd quadrant 2-3 times
1. Peptone digest of animal tissue 5 g/L  
 Touch 4th quadrant once only
2. Beef extract 1.5 g/L  
Do not remove petri dish cover, open it at 30 degree angle only
3. NaCl 5 g/L
4. Agar 15 g/L
5. Sterile H2O 1L
 Original sample is diluted several times to reduce microbial population
 sufficiently to obtain separate colonies upon plating
 STEPS:


 get 1 ml of 90% normal saline solution
After dilution is complete,
(NSS) & add in petri dish
 
 Pour cooled melted agar
 
Wait for melted agar to solidify

EXERCISE 6
Inhibition and Destruction of Microorganisms by Physical Agents

* Disinfection – kill microorganisms except spores

* Sterilization – kills microorganisms including spores
II. 4 Quadrant Streak Plate Method
PHYSICAL METHODS OF STERILIZATION
  By streaking, dilution gradient is established on surface of plate
1) INCINERATION
 USES: nutrient agar
  - 870-980C
 Solidified (opaque) - burned to ashes (ie: bacteriologic instruments)
 
 Liquefied (darker) - most common, simplest, fastest
  - not used bc of toxin air emissions/ presence of heavy metals
Incubated for 24-48 hours

2) MOIST HEAT
- sterilize biohazardous trash, glasswares, oil petrolatum/powder
Page 5 of 21
 BACTERIOLOGY I || Megan Arceño | Cloie Casinillo | Angela Magsucang A.Y. 2015 – 2016, 1st semester

- Bacillus stearothermophilus (sporing)

> Autoclave = - 121C at 15 psi for 15-20 mins B. Phenols
> Principle: Steam under pressure 1) Phenols – disinfect blood, feces, mucus, sputum
> 134C for 3 mins (Flash Autoclave) 2) Cresol (Lysol w/ soap) – disinfect inanimate objects
> 132C for 30-60 mins (treating infectious med wastes) 3) Substituted phenol – added to surface active disinfectants to increase
their germicidal action
3) DRY HEAT 4) Alcohol – 50-70% effective concentration
- 160-180c for 1 ½ - 3 hours
- requires longer exposure time II. Agents that denature protein
= causes unfolding of polypeptide chains
4) IONIZING RADIATION i. Acids and alkalies
- Sterilize syringes, catheters, gloves ,etc. before use ii. Alcohols
- Hydroxyl radical will react w/ the cellular components of cell (DNA)
III. Agents that modify functional groups of protein & nucleic acids
5) FILTRATION 1) Heavy metals (ie. 1% AgNO3 – eyes of newborn to prevent
- Does not kill microorg, rather filter them Neisseria gonorrheae/ Opthalmic neonatorum)
- Method of choice for antibiotic solns, toxic chemicals, vaccines 2) Oxidizing agents
- for heat-sensitive materials - Chloride (swimming pool treatment)
> Filtration of liquid - pulling soln across a membrane - Iodine (disinfect skin before surgery)
- Cellulose acetate, Cellulose nitrate 3) Hyrogen peroxide – penetrate; and act as anaerobes
> Filtration of air – HEPA filters 4) Dyes – active: Gram positive organisms
5) Alkylating agents
a) Ethylene oxide – most common chemical sterilant used in
gaseous form sterilizing heat sensitive objects
b) Formaldehyde vapors and vapor H2O2 – For HEPA filters
PHYSICAL METHODS OF DISINFECTION c) Glutaraldehyde – kills spores (3-10 hrs); sterilize bronchoscopes
1) BOILING at 100C for 15 mins – kills vegetative bacteria d) Peracetic acid - sterilize surgical instruments

2) PASTEURIZATION – kills food pathogens

> Classic pasteurization = 63C for 30 mins PROCEDURE PROPER
> High temperature, short time pasteurization = 72C for 15 secs * Filter paper squares impregnated w/ Bacillus subtilis &
E.coli 1) Control
3) NON-IONIZING RADIATION 2) Boiling (30 mins)
- Thymine dimers inhibit replication of microorg 3) Water bath adjusted to 80C (30 mins)
- ie: UV light 4) Autoclaved
5) Hot air

CHEMICAL METHODS OF STERILZATION AND DISINFECTION * Incubate at 37C

I. Agents that damage the cell membrane
= disrupts structural organism of mem. interfering w/ normal func Control Boiling 80C Hot air Autoclave
oven
A. Surface Active Disinfetants Bacillus Turbid Turbid Turbid Clear Clear
1) Soap & detergents = remove organisms through scrubbing subtilis
2) Benzalkonium chloride = 1:1000 aq. soln. kills vegetative bacteria in 30 mins Escherichia Turbid Clear Clear Clear Clear
except Mycobacterium TB coli
^^ More effective to Gram positive microorganisms
Page 6 of 21
 BACTERIOLOGY I || Megan Arceño | Cloie Casinillo | Angela Magsucang A.Y. 2015 – 2016, 1st semester

► BROTH used = NUTRIENT BROTH

► Control = Nutrient broth + impregnated filter paper w/ bacteria PROCEDURE PROPER
= TURBIDITY (+)/ presence of bacteria * 1ml E.coli + 10 ml Disinfectant
► Evidence of growth = TURBIDITY * After intervals of 5, 10, 15, 30 mins, transer an inoculum (5 loopfuls) from each mixture
►BACILLUS SUBTILIS (sporing streptobacilli) to the Lactose Broth Tubes
- Only killed w/ STERILIZATION= killed with HOT AIR & AUTOCLAVE * Positive control – get directly from the bacterial suspension and add directly to
- Interpretation = Bacteria were killed in hot air and autoclave the LBTw/DFT
► ESCHERICHIA COLI (non sporing coccobacilli) * Incubate at 37C for 48 hrs
- Killed w/ BOILING, 80C, HOT AIR & AUTOCLAVE
- Interpretation = Bacteria were killed in boiling,
80C, hot air and autoclave
> Turbid = not killed EXERCISE 8
> Clear = killed Antimicrobial Susceptibility Testing
* If control is clear, invalid
The Kirby Bauer test - routinely used to determine susceptibility or resistance of a pathogenic
organism in various antimicrobial agents
EXERCISE 7
> PRINCIPLE: A standardized suspension of organisms is inoculated on Mueller-Hinton agar.
Action of Disinfectants on Bacteria Paper disks impregnated with specific antibiotic concentrations are placed into the agar. After
18-24 hours of incubation, the diameters of the zones of inhibition are measured. The results
* Study bactericidal effect of concentration and time of exposure of disinfectants in bacteria are compared with established values to determine the organism’s susceptibility or resistance
* To detect relative effectiveness of a certain standard commercial disinfecting agents to each antibiotic.

► Test organism = ESCHERICHIA COLI Antimicrobial Susceptibility Testing – performed only after positive identification of
► Culture medium = LACTOSE BROTH W/ DURHAM’S FERMENTATION TUBE microorganism by biochemical testing
* Peach to Hot pink (if there is growth)
Indications for performing Antimicrobial Susceptibility Testing:
► INDICATION OF GROWTH  Determine sensitivity & resistance of bacteria to antimicrobial compounds
* one/ two/ all observed = growth of organism 
 Measure susceptibility of bacteria
1) Change in the pH medium
= change of color in medium METHODS:
* Indicator - Andrade’s indication (peach to hot pink)
= organism ferment lactose to lactic acid (lower pH/acidic) I. BROTH DILUTION/ AGAR DILUTION
2) Gas Formation in the Durham’s fermentation tube a. Quantitative
= presence of Hydrogen gas in the DFT traps gas that is formed b. Determines:
3) Turbidity i. Minimum Inhibition Concentration (MIC)

> Disinfectants used:  what concentration of bacteria is inhibited


1) 5% phenol  inoculate to antimicrobial free media = turbidity (+)
2) 1:1000 mercuric chloride 
3) tincture of iodine ii. Minimum Bactericidal Concentration (MBC)
4) 70% alcohol
 concentration that can kill 99% of growth
5) 3% H2O2
6) Lysol
*indication of MCB: ABSENCE OF GROWTH
> Time Interval: 5, 10, 15, 30 minutes
> Incubation temp: 37C
Page 7 of 21
 BACTERIOLOGY I || Megan Arceño | Cloie Casinillo | Angela Magsucang A.Y. 2015 – 2016, 1st semester

II. DISK DIFFUSION o For N. gonorrheae

 
a. Qualitative (indirect method)  Petri dish size
b. Kirby – Bauer Diffusion (since 1966) o 150 mm (60 mL: 10-12 discs)
o 100 mm (25 mL: 5-6 discs)
II b. KIRBY – BAUER DISK DIFFUSION METHOD - the presence or absence of growth o 15 mm space from edge
around the disc is an indirect measure of the ability of the antibiotic to inhibit the o 4-6 mm standard agar depth
microorganism
<4 mm: false susceptible result
 Uses antibiotic impregnated disc

  >6 mm: false susceptible result
 Zone of Inhibition (ZOI) – indicates inhibition of bacteria
 
 Vernier caliper – reads the ZOI
o Cation content:
 
 Susceptible – antibiotic kills bacteria
 Ca & Mg too high:
 
 Resistant – antibiotic can’t kill bacteria  
 False resistance
 
 Inoculum suspension – bacterial suspension  
   Tetracycline & aminoglycosides
DIRECT COLONY SUSPENSION
 Ca & Mg too low
o NSS (dilutor)  
   False sensitive
 18-24 hours  
P. aeruginosa

Leave 
15 minutes in NSS o pH
o Prepared standard
    7.2 – 7.4
 Absorbance reading: 0.08 – 0.13
    <7.2 (false resistance)
 Optical density: 625 nm
 
 >7.4 (false sensitive)
 CELL DENSITY: 1.5 X 108 CFU/mL  
  Inoculation of MHA
 Good for 6 months use only
 
o STREAK: OVERLAPPING STREAK METHOD
 used when making bacterial suspension
 
1. dip, streak swab into inoculum
made thru comparing with:
2. rotate 60 degrees after streaking
McFarland Standard =
3. streak, rotate again to 60 degrees
0.5 mL of 1.175% BaCl2 + 99.5 mL of 1% H2SO4 4. rim 2x

  o let inoculum sit on plate for 3-5 minutes and not more than 15
LOG PHASE GROWTH minutes
o Broth culture media
o Incubate: 2-6 hours before use *ANTIBIOTIC DISK PLACEMENT (Automatic method)
o Mueller – Hinton Agar  
  Technical errors
 For non-fastidious organisms
o False resistance
o False sensitive
 
 Streptococci (MHA w/ 5% sheep blood)
 
 
  
  Heavy inoculum Light inoculum
Incubated at 5-7% CO2
   
    Thick medium  Thin medium
Methicillin-resistant, MRSA,
S. aureus (MHA w/ 2% NaCLl) 

Delay in disc application
 
  Prolonged 
Medium base factors:  incubation time
o X (hematin) Increase Ca/Mg
o V (NAD)  P.
(aminoglycosides)
aeruginosa
   
GC (Gonococcal) agar Lack of incubation time
Page 8 of 21
 BACTERIOLOGY I || Megan Arceño | Cloie Casinillo | Angela Magsucang A.Y. 2015 – 2016, 1st semester

 
AFTER 16-24 HOURS/ AFTER INCUBATION PERIOD Biochemical tests
   I.Pigment production
Check confluent lawn of growth
    
 Check purity of growth Agar in slanted medium
 
  Has a yellowish white color when uninoculated
ZOI: round off to nearest mm
II. Catalase test
o If overlapping: measure radius & multiply by 2  
   Done on Trypticase soy agar
 Vernier caliper  
 Prepared by slant = slanted position
1. Main scale (in inches or cm) 
Has a yellow color when uninoculated

2. Vernier scale III. Hemolytic pattern and morphology
3. Inside jaws (inner diameter)   
Uses blood agar
4. Outer jaws (measure the ZOI) 
Ideal RBC added in preparation of blood  agar is sheep blood since
5. Depth probe it provides better results in hemo pattern
  IV. Mannitol fermentation test
 Non-viable cells – organism is dead  
 
 Run on mannitol salt agar
 Interpretation of results  
 Prepared in plate
o (S) sensitive – microorganism is killed, likelihood of therapeutic success  
 Has a faint orange color when uninoculated
o (I) intermediate – bacteria inhibited in vitro, uncertain therapeutic effect V. Coagulase test
o (R) resistant – bacteria inhibited in vitro, high likelihood of 
therapeutic failure 
Best biochemical test that differentiates S. aureus from S.
epidermidis
   
 Resistance (caused by) CATALASE TEST
o Enzymatic degradation/ modification of antimicrobial agent o Reagent: H2O2
o Decrease in uptake of antimicrobial agent o Test that demonstrates ability of organisms to reduce H2O2 to H2O and O2
o Inoculate 3-5 loopfuls on the trypticase soy agar
o Altered antimicrobial target
  o Catalase-producing organisms = resists H2O2 disinfectant
 TEST MICROORGANISM: PROTEUS MIRABILIS o Catalase (+) = staphylococcus – bubbles formed o
 
 Pseudomonas aeruginosa: may cause green pigment on plate Catalase (-) = streptococcus – no bubbles formed
 
 Zone of inhibition:  
o Double zone: measure innermost zone COLONIAL MORPHPLOGY
o Repeat test: measure colony forming zone o Streaking method: 4-QUADRANT STREAK PLATE METHOD
o Incubate @ 37 degrees C for 24 hours
o Zone with swarming: disregard swarming, measure obvious zone, measure  
pigment of S. aureus is best demonstrated at this temperature
the obvious demarcation
 
 ETEST (uses plastic strips; disc differentiation + MIC data) o S. aureus
    
BIOMIC (disc differentiation + video digital analysis) smooth, mucoid, golden yellow colonies
  
produces: yellow pigment (lipochrome)
o S. epidermidis
  
rough, round, white colonies
 
produces: white pigment (no name)

EXERCISE 9
 
Staphylococcus HEMOLYTIC PATTERN
o Streaking method: 4-QUADRANT STREAK PLATE METHOD
  Species

o Incubate @ 37 degrees C for 24 hours
   o Culture medium: Blood agar
aureus – most common
     
epidermidis – part of the normal flora of the skin Enrichment and differential medium
 
 saprophyticus – causes UTI in women  Enrichment: supports growth of all org
 
intermedius – from dog bites  Differential: diff hemolytic patterns
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BACTERIOLOGY I || Megan Arceño | Cloie Casinillo | Angela Magsucang A.Y. 2015 – 2016, 1st semester

o Growth of organisms on blood agar is dependent on organisms ability o S. aureus: COAGULASE (+) clumping
to utilize haemoglobin in blood agar o S. epidermidis: COAGULASE (-) no clumping
o Hemolytic pattern caused by organism’s ability to destroy
hemoglobin Summary of results:
o Brown’s classification STAPHYLOCOCCUS
   STREP
Beta hemolysis aureus epidermidis sapro
  complete destruction of RBC in BAP No bubble
Catalase test Bubble formation (+)
 BAP turns yellow formation (-
   )
Alpha hemolysis
  Partial destruction of RBC in BAP -smooth, mucoid, - rough, round, white
 BAP exhibits greenish-brown discoloration Colonial golden yellow colonies colonies
--- ---
   morphology -yellow to deep yellow - gray to white pigment
Gamma hemolysis
pigment (lipochrome) (no name)
 Absence of destruction/ no destruction of RBC in BAP Haemolytic
 Red color remains in BAP pattern
Beta hemolysis (yellow) Gamma hemolysis (red) ---
o S. aureus: BETA HEMOLYSIS Mannitol  
o S. epidermidis: GAMMA HEMOLYSIS fermentation Orange yellow (+) Orange pink (-) ---
  test
MANNITOL FERMENTATION TEST Coagulase
Clumping (+) No clumping (-) ---
o Uses a sugar/carbohydrate: mannitol production
o Mannitol salt agar Novobiocin
--- Susceptible Resistant
   sensitive
Selective and differential medium
 Selective: inhibits growth of Gram (-) organisms &
selects growth of G (+) organisms only; allows growth
 of organisms that can tolerate high levels of salt only
EXERCISE 10
 Differential: differentiates (+) fermentation from (-
Beta Hemolytic Streptococcci
 ) fermentation
  
Has a faint orange color
  
7.5% NaCl
  Group Species Pattern of Hemolysis Other info
 Tests ability of organism to ferment mannitol
  A S. pyogenes Beta hemolytic
 Has a neutral pH (7.0)
  B S. agalactiae Beta hemolytic
Indicator: Phenol red = acid products (from fermentation)
o S. aureus C S. equisimilis Beta hemolytic
 
N/A
 (+) fermentation
S. equi Beta hemolytic
    S. zooepidemicus Beta hemolytic
Change in pH (orange yellow)
o S. epidermidis S. dysagalactiae α/ γ hemolytic
  
(-) fermentation D S. faecalis α/ γ hemolytic Enterococci
   S. faecium α/ γ hemolytic Resists 6.5 % NaCl
Change in pH (orange pink)
  S. durans α/ γ hemolytic Grows very well in salt medium
COAGULASE PRODUCTION S. bovis Non- enterococci
o PRINCIPLE: ability of organism to clot fibrinogen α/ γ hemolytic Sensitive to 6.5 % NaCl
o REAGENT: plasma
Cannot grow in salt medium
o Types

 Slide coagulase: uses slide; detects  bound coagulase (bound Species used
coagulase is known as clumping factor)   
  S. pyogenes
Tube coagulase: uses tube; detects free coagulase  
S. agalactiae

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BACTERIOLOGY I || Megan Arceño | Cloie Casinillo | Angela Magsucang A.Y. 2015 – 2016, 1st semester

 
  o
Microscopic morphology  protein that acts together with
Diffusible heat stable extracellular
Cocci in chains the Beta hemolysin of S. aureus
 o Both species above present the same morphology o Culture media: BLOOD AGAR PLATE (BAP)
  
  Procedure
LANCEFIELD CLASSIFICATION  Streak center of BAP with S. aureus

o Most reliable classification of Streptococci; Brown’s classification  Streak the Beta – haemolytic streptococci
can’t provide accurate classification perpendicular to the S. aureus w/o touching it
o Specific antigenic group: C-wall polysaccharide o GROUP B: produces CAMP factor
 antigen found on outer cell wall of streptococci o GROUP A & B: produces enhanced Beta – hemolysis in the junction called
o Classifies based on C-wall; differentiates different C-walls ARROW HEAD HEMOLYSIS
o Classifies according to letters of alphabet o POSITIVE: Streptococcus agalactiae
  o NEGATIVE: Streptococcus pyogenes
BACITRACIN DISC SENSITIVITY TESTING
o Identifies group A strep from group B strep
o 1 organism is sensitive and 1 organism is resistant
  group A (sensitive/susceptible) w/ ZOI
 group B (resistant) w/o ZOI
o Uses OVERLAPPING STREAK METHOD
EXERCISE 11
o Incubate @ 37C
Alpha Hemolytic Streptococci
o Principle
 Demonstrates the ability of Bacitracin disk to inhibit the growth Species used
of group A streptococci and promote the growth of group B   
S. pneumoniae
streptococci  
Veridans streptococci
o 0.04 units of Bacitracin is used in the experiment 

  Low concentration  
Morphology
 inhibits group A only
o Both present diplococcic morphology
 o S. pneumonia (encapsulated)
  o Veridans streptococci (non-encapsulated)
VIRULENCE FACTOR
o For Group A streptococci   HISS STAINING METHOD

 Hemolysins (capable of producing beta hemolysis) o Modified of the Anthony’s Method
 Streptolysin O (SLO) o Demonstrates capsule of S. pneumoniae
o Oxygen labile
o Easily destroyed by oxygen   OPTOCHIN DISC SENSITIVITY TEST

 Streptolysin S (SLS) o Presumptive test for ID of S. pneumoniae
o Oxygen stable o Also called
o Not easily destroyed by oxygen   
P – disks (6mm)
   
Do not forget to STAB the Blood Agar Plate  Ethyldrocupreine hydrochloride
 o Culture media: BAP
o For oxygen to enter the petri dish oxygen gets trapped at bottom of
 o OVERLAPPING STREAK PLATE METHOD
petri dish after pouring bacterial mixture bacteria is visible
  o RESISTANT: Veridans streptococci
CAMP test (Christie, Atkins, Munch, Peterson) o SENSITIVE: S. pneumoniae
o Presumptive test in the identification of Group B streptococci 
o Demonstrates CAMP factor of Group B o (+) result: Greenish brown disc measure ZOI

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 BACTERIOLOGY I || Megan Arceño | Cloie Casinillo | Angela Magsucang A.Y. 2015 – 2016, 1st semester

 
BILE SOLUBILITY TESTING
o (+) result – clearing of bacterial suspension III. Determine manifestations of patient for that specific infection
o Principle o N. gonorrheae
   
Foul-smelling vaginal discharge/ foul smelling urethral discharge
Makes use of bile salt 20% sodium deoxycholate/ sodium  
taucholate/ sodium dodecylsulfate  Anuria (most severe)
   
Compared w/ 1 McFarland Standard Burning sensation during urination
 Heavier turbidity than 0.5 McFarland o N. meningitidis
o S. pneumonia   
Seizure (most severe, results to brain damage)
   
Contains intracellular autolytic enzyme calledAMIDASE  Headache
capapble of destroying the organism automatically 
Light headedness

o CONTROL: 5 drops NSS
o EXPERIMENTAL: 20% sodium deoxycholate (5 drops)

Cause activationof amidase & clearing of bacte suspension o IV. Identification & isolation of species through culturing
BILE SOLUBLE (+): S. pneumonia o Culture media
o BILE INSOLUBLE (-): Viridans streptococci   
Thayer Martin Agar
  not specific to Neisseria
 TMA is Mueller Hinton Agar with 5% RBC enriched w/
haemoglobin
EXPERIMENT 12  
 MODIFIED THAYER MARTIN AGAR
Neisseria
  Culture media of choice
Species used  Modification: addition of antimicrobials (V-C-N-T)
   o V – vancomycin (inhibits gram neg org.)
N. gonorrheae
 
o C – colistin (inhibits gram pos org.)
N. meningitidis
 o N – nystin ( inhibits growth of fungi)
 o T – trimethoprim (inhibits swarming proteus)
Investigation steps:
 V-C-N-T acts as inhibitors
I. Morphology  
 CHOCOLATE AGAR
o Gram (-) appearing coffee bean-shaped
 Alternative culture media
o Commensal organism (when bacte enters body, only bacte benefits)
o Strongly oxidase positive

o Incubate @ 37C for 24H in candle jar because Neisseria is anaerobic
o Catalase positive
V. Oxidase Test
o Diplococci
  o Presumptive test to confirm that Neisseria species was the
 Intracellular bacteria isolated and not a different species

 Found in cytoplasm eaten by WBC as WBC fights it during o Identifying enzyme: CYTOCHROME OXIDASE
 bacterial invasion
o Reagent: 1% para – aminodimethylaniline oxalate (redox
 Indicates current/ active infection reagent) o REACTION:
    
Extracellular Pink (initial/orig color) maroon (after 10s) dark red (after
 
 Found outside the cell not eaten by WBC 20s BLACK (after 30s—color end product)
 Indicates patient on therapy/ undergoing treatment  N. gonorrheae & N. meningitides = both are (+) for oxidase test

II. Determine type of infection organism produces VI. Gram staining
o N. gonorrheae: sexually transmitted Infection o To confirm morphology as diplococci
o N. meningitidis: infection involving CNS
i. Meningitis
ii. Meningitidis
iii. Waterhouse-Friderichsen syndrome
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 BACTERIOLOGY I || Megan Arceño | Cloie Casinillo | Angela Magsucang A.Y. 2015 – 2016, 1st semester

VII. Carbohydrate fermentation test o Overt/ true pathogens

o Also known as: SUGAR FERMENTATION TEST   Causes infection in GIT (thus, organisms of interest)
o Culture media: CYSTINE TRYPTICASE/ TRYPTIC AGAR (CTA)  Transmission: ORAL-FECAL ROUTE
o Sugars used: Glucose, Lactose, Maltose, Sucrose
o Uninoculated culture media Investigation steps:
 Orange in color I. Specimen collection
   
Has a pH of 7.0 PHENOL RED (pH indicator)  STOOL/ FECES sample: specimen of choice
o By product: always acid  Not sterile (has contaminants wherein other species of
o Results: (+) YELLOW, ACIDIC ; (-) PINK, ALKALINE bacteria can grow), thus, ENRICHMENT MEDIA is used

o Results  Growth on enrichment media indicates successful growth
of overt organisms
Species Glucose Maltose Lactose Sucrose
N. gonorrheae + - - - II. Transport Media
N. meningitidis + + - -   
Carey Blair medium (most common)
N. lactamica + + + -   
Modified Stuart medium (rectal swab)
N. sicca + + - +   
Glycerol M phosphate (fecal specimen)
 
Stuart Medium

III. Enrichment media – enhance the growth of Gram (-) organisms

Tetrathionate broth (18-24 hrs.)
EXERCISE 13 + bile salts – S. typhi, (specific for) Shigella sp.
Enterobacteriaceae + brilliant green (1:100,000) – Salmonella sp.
Selenite F broth (12-16 hrs.)
  Collectively known as: ENTERICS

- Shigella sp., Salmonella
 
 Are gram negative rods Hajna GN (Gram Negative) broth
  
Capsule: Klebsiella
   IV. Plating media
Slime layer: Escherichia
   Slightly Selective = all Gram ( -)
Most have fimbriae and it has complex cell wall
   Moderately Selective = Salmonella & Shigella
None produce cytochrome oxidase: oxidase- negative
   Highly Selective = Salmonella
Variety of lactose fermented
  
Biochemical testing: only test to differentiate Enterobacteriaceae species
  (SS) MacConkey
2 kinds:
  
o Opportunistic pathogens Sugar: lactose
  Enterics that are part of normal flora in human’s GIT  
Indicator: Neutral red (pH indicator)


  Doesn’t cause infection 

Inhibits gram positive bacteria with bile salts & crystal violet


  GIT serves as habitat Differential: Lactose fermenter – pink colonies
  Causes infection outside of GIT only Non-lactose fermenter – colorless colonies
 
 Pneumonia (in lungs)  Uninoculated agar: LIGHT PINK
 
  Meningitis (CNS) Differential: diff between lactose fermenters & non-lactose fermenters

  UTI (urinary system)  

 Septicemia/ sepsis (blood) MacConkey Sorbitol Agar

 Examples: o Differentiates VTEC from other E. coli serotypes
Serratia Enterobacter Escherichia o VTEC (with Verotoxin) ferments sorbitol easily o
Other E. coli serotypes do not ferment sorbitol o
Klebsiella Citrobacter Morganella
Proteus Providencia

E. coli O 157:H7 causes haemolytic urine
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 BACTERIOLOGY I || Megan Arceño | Cloie Casinillo | Angela Magsucang A.Y. 2015 – 2016, 1st semester

Non-lactose fermenter – colorless colonies

(SS) Eosin-methylene blue agar

 
 
 Sugar: lactose & sucrose Uninoculated: ORANGE
  
Indicator: eosin y & methylene blue (known as: ANILINE DYES)
 
Inhibits gram positive bacteria with aniline dyes (gram positive inhibitor)

Differential: Lactose fermenter – dark colored colonies (MS) Hektoen-Enteric agar
Non-lactose fermenter – colorless colonies  
  Sugar: lactose, sucrose, salicin
 E. coli = greenish metallic sheen
   Indicator: bromthymol blue, acid fuchsin (for pH)
Uninoculated agar: DARK RED/ MAROON
Ferric ammonium citrate & Sodium thiosulfate ( for H2S)
 Differential: production of H2S
No production of H2S
 Colonies: Lactose fermenter – salmon pink to orange yellow colonies
Non-lactose fermenter – greenish blue colonies
- Greenish blue color is due to culture media’s color
 
Uninoculated: GREEN

(MS) Salmonella Shigella Agar (SSA)

 
Sugar: lactose
 (MS) Xylose-lysine desoxycholate
Indicator: Neutral red & bromthymol blue (for pH)
  
Ferric ammonium citrate & Sodium thiosulfate ( for H2S) Sugar: lactose, sucrose, xylose
  
Inhibits other organisms other than Salmonella-Shigella  Xylose makes culture media specific for Shigella since it is
 
 Contains high concentration of bile salts & sodium citrate  the only Enterobacteriaceae that doesn’t ferment xylose
- Bile salts inhibit normal Gram (-) organisms  Lysine supports non-lactose fermentation of Salmonella
 Differential: Salmonella – produce H2S (colorless w/ black center)  Indicator: phenol red (pH)
Shigella– doesn’t produce H2S (colorless w/o black center) Ferric ammonium citrate, sodium thiosulfate (H 2S)
 Colonies: Lactose fermenter – pink colonies  Inhibitor: Sodium deoxyxcholate
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 BACTERIOLOGY I || Megan Arceño | Cloie Casinillo | Angela Magsucang A.Y. 2015 – 2016, 1st semester


Colonies: Lactose fermenter – yellow to orange
Non-lactose fermenter – red colonies
w/ H2S formation—Salmonella
w/o H2S formation—Shigella
 
Uninoculated: BRIGHT RED

  Enterobacter aerogenes

  
Enterobacter cloacae
* EMB = “fish eye” (pink mucoid w/ central dark colored
colonies) > capsulated
> aerogenic = gas producing organism (needs oxygen for
survival) > Lysine Decarboxylase
(+) = Enterobacter aerogenes
(HS) Bismuth Sulfite Agar (-) = Enterobacter cloacae
  
Sugar: glucose
  
Indicator: bismuth sulphite, ferrous sulfate (H2S)

 Specific: Salmonella typhi – jetblack colonies with metallic sheen; Other organisms – green,
brown or brown black colonies
 
Uninoculated: YELLOWISH GRAY

 
Proteus/ Proteae
A. Swarming (spreading) proteae
o flagella (highly motile) = spreading/
concentric ring growth
o UTI = kidney stoned (called: staghorn
V. Colonial Morphology calculi = due to urease enzyme)
o Presumptive ID of bacteria ► Proteus vulgaris
   ► Proteus mirabilis
Escherichia coli B. Non swarming proteae
 
 Citrobacter freundii ► Morganella morganii (small, pinpoint, pink colonies)
* EMB = dark purple colonies with greenish metallic sheen ► Providencia rettgeri
* MAC = flat, dry, pink colored colonies
  Klebsiella pneumoniae

  
Klebsiella oxytoca
* EMB & MAC = large, mucoid (encapsulated), pink colonies
* String test = positive (confirms mucoid characteristic of org)
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 BACTERIOLOGY I || Megan Arceño | Cloie Casinillo | Angela Magsucang A.Y. 2015 – 2016, 1st semester

 Differentiates: lactose/non-lactose fermenter; H 2S production

  Serratia marcescens

 Indicators: Phenol red (pH),
* EMB & MAC = large, mucoid (encapsulated), red colonies (prodigiosin) Ferrous sulfate & sodium thiosulfate (H2S prod)
Prodigiosin = red pigment (room temp)  
Sugars: 0.1% Glucose (found on butt)
= Non-water soluble pigment 1% Lactose & 1% Sucrose (found on slant)
= Cotton swab is used to absorb colonies (confirm red)   
Reaction:
Arabinose 1) Sugar fermentation (*all entero ferments glucose*)
(+) Serratia marcescens > yellow = fermented
(-) Enterobacter > pink = not fermented

Ac (yellow) Al (pink) Al (pink) Al (pink)

Ac (yellow) Al (pink) Ac (yellow) Ac (black)

2) H2S production (Cysteinase enzyme- responsible for production)

3) Gas production
- splitting of medium (division of butt/slant)
- single gas bubble in the medium
- complete displacement of medium from bottom of tube
- slight indetation of medium from side of the tube
IMVIC TEST: INDOLE-METYHL RED-VOGES PROSKAUER-
CITRATE A. Indole
  Salmonella typhi
 * Indole production = Tryptone broth (clear, light yellow)
 
 Contains Tryptophan (Tryptophanase – enzyme prod by org)
* BSA= jet-black colonies (production of H2S) colonies w/ metallic sheen (silver)  
Indole is produced (by addition of Kova’c/ p-dimethylaminobenzaldehyde)
Typhoid fever = Widal test (diagnostic test)
= purple ring (+); no ring (-)
  Pseudomonas aeruginosa
 B. Methyl Red
* Swarming colonies; emit grape-like odor or corn/ tortilla-like odor * Detect acid products (prod by mixed acid fermentation) with addition of methyl red
> Non-fermentative bacilli (cannot ferment sugars) * Culture medium: MR-VP broth (do not contain pH indicator)
>causes NOSOCOMIAL INFECTION (community-acquired/ hospital-acquired infxn) = uninoculated: clear yellow
> Produce pigments: * pH indicator: Methyl red
1. Pyocyanin (turquoise / blue-green colored pigment) > (+) = turbid, red
= water soluble & chloroform extractable > (-) = turbid, yellow
= best isolation media: Pseudomonas P agar/ King’s A medium
2. Pyoverdin/ Fluoroscein (yellow green pigment) C. Voges-Proskauer
= water soluble & not chloroform extractable * Detect Acetoin (Acetyl Methyl Carbinol) & Butanediol in the presence of KOH and
= best isolation media: Pseudomonas F agar/ King’s B medium alpha-naphthol (KOH & a – naphthol are reagents = responsible for red color)
3. Pyorubin (red pigment) * Culture medium: MR-VP broth (do not contain pH indicator)
4. Pyomelanin (brown pigment) : uninoculated: clear yellow
* Exposure to oxygen = accelerated VP test (+) = red (–)
= yellow
VI. Biochemical tests D. Citrate Utilization Test
 * Utilization of citrate (sodium citrate) as carbon source
TRIPLE SUGAR IRON (TSI AGAR) prepared in test tube * Culture medium: Simmons Citrate Agar
  : uninoculated: green
Uninoculated:orange
Slant – aerobic; Butt – anaerobic * indicator: Bromthymol blue (+) = blue
(–) = green
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 BACTERIOLOGY I || Megan Arceño | Cloie Casinillo | Angela Magsucang A.Y. 2015 – 2016, 1st semester

MYCOBACTERIUM
SIM TEST: SULFITE INDOLE MOTILITY TEST   
Obligate aerobic organism
* Detect motility (main objective)  
Nonmotile; nonsporogenous; non encapsulated

* Culture medium: SIM medium = semi-solid   
Highly pathogenic
* Other reactions:  
Mycolic acid (responsible for acid fast characteristic)
1) Motility
o Acid fastness best demonstrated (method) : Cold Kinyoun, Acid
2) H2S Fast staining
3) Indole production = purple ring after adding Kovac’s reagent o M. acid enables resitance to decontaminating agents and drying
* Preparation in butt:   
Does not stain readily w/ basic aniline dyes
(+) = haziness = motile  
 71 species classified accdg:
(–) = clear = non-motile
o EPIDEMIOLOGY & ASSOCIATION w/ DISEASE (basis)
o M. tuberculosis complex
 
M. tuberculosis, M. bovis, M. africanum
UREASE TEST
 M. bovis BCG : vaccine against PTB
* Detect urease
o Non tuberculous mycobacteria (NTMs)
* Breakdown of urea to ammonia (basic) + CO2  
 Mycobacteria other than tubercle bacilli
* Resulting ammonium carbonate is identified as light/dark pink  
Doesn’t cause tuberculosis
* Culture medium: Christensen’s Urea Broth (main substance is urea)
o M. avium comples : in patients infected with HIV/ AIDS
: uninoculated: orange
o M. leprae : non cultivable, grown only in foodpads of armadillos
* pH indicator: Phenol red  
 RUNYON CLASSIFICATION
* reactions: (+) pink – urease produced
o Classifies accds : rate of growth, colonial pigmentation
(–) yellow – no urease produced
o Grouping
I.Photochromogen (visible in artificial CM after 7 days)
II.Scotochromogen (pigment seen in light and dark, growth after 7
Slow growers
REDUCTION OF NITRATES TO NITRITES
days incubation)
III. Non photochromogen (growth after 7 days)
IV. Rapid growers (growth < 7 days)
PHENYLALANINE DEAMINATION MYCOBACTERIUM TUBERCULOSIS
TEST * Deamination of phenylalanine  
 Founded : Robert Koch
Yields: Phenyl pyruvic acid + 10% FeCl2  
 Metachromatic granules : Much granules (where org derives energy)
(+) green

(–) brown
  due to hydrophobic cell nature and clumped growth o Slow
Resistance to chemical agents
* Culture medium: Phenylalanine agar growth (responsible)
o Clumping = nutrients not easily allowed into cell
o Slow growers : 2-60 days after incubation
  
FERMENTATION OF CARBOHYDRATES (CHO) Causes pulmonary TB : person-to-person inhalation of droplets (airborne)
  
* Stab butt in inoculation (90 degrees, halfway) Collection : deep breath 3x then cough, empty area, oppose direction of wind
 
* Culture medium: Cystine Trypticase Agar (CTA) + Sugar Diagnostic lab test

* pH indicator: Phenol red o Biosafety Cabinet Level II (prevent transmission of bacte)

(+) yellow = fermentation o Mantoux test (Tuberculin Skin Test)
(–) pink = no fermentation   To identify infxn of TB
* Presence of gas noted by crack in medium (extends sidewards from the line of stabbing)  Principle : antigen –antibody reaction
o Antigen : purified protein derivative
 
Purified M. TB

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BACTERIOLOGY I || Megan Arceño | Cloie Casinillo | Angela Magsucang A.Y. 2015 – 2016, 1st semester

 Induration (swelling) in site of injection o Usual culture media used for ID : 1 liquid, 2 solid mediums
 
 o > 10 mm (+) TB infxn Niacin test : differentiates M. tuberculosis from M. leprae
 o > 15 mm – px infected w/ HIV o Niacin test (+) M. tuberculosis
 Nonspecific test for TB o Niaci test (-) M. leprae
 
Mycobacteria growth indicator tube (MGIT) system
o Direct Sputum Smear o Modified 7H9 broth
  Confirms Mantoux test o Orange fluorescence (+)
  Red bacilli against blue background
 Auramine-Rhodamine stain : best demonstates MYCOBACTERIUM LEPRAE
 
o Gastric washing : for patients that can’t produce sputum sample Morphology : parallel bundles/ globular masses (globi) = PACKETS OF CIGAR
 
such as pediatric patients Specimen of choice : skin scrapings
o CSF WATER BACTERIOLOGY
 most commonly accepted specimen for TB   
 Bacterial examination of water
 
 if specimen is from CSF, dse associated with it is called  Determines potability of two samples from different sources
tuberculous meningitis   
 Identify presence/ absence of E.coli & other coliforms in water sample
 Pellicle (cobwebs) formation : characteristic of tuberculous 
meningitis (if spec in placed in fridge overnight)   o
Potability (characteristic of water if safe for consumption)
potable : safe for consumption; uncontaminated
o Limulus Lysine test : detect gram (-) bacilli in M. tuberculosis o nonpotable : unsafe for consumption; contaminated
o Routine sample : sputum sample (constantly decontaminated)  
Intestinal pathogens
 Gold standard : N-acetyl-L-cysteine (NALC) & NaOH o Salmonella typhi : typhoid fever
 o NALC (mucolytic agent, liquefies sputum) o Shigella dysentery : bacillary dysentery
o NaOH (toxic decontamination agent, removes   
Fecal contamination (pathogenesis) : how contamination spreads
 other bacteria) 
No direct test can identify
presence of intestinal pathogen in the water sample, thus an
 Heat in between quadrants to remove contaminants from indicator organism is used
 the organism 1. Escherichia coli
* do not heat CSF = heating kills organism   
Indicator organism for contaminated water because:
   Most abundant gram (-) enteric organism in GIT of
Culture media warm-blooded organisms
o Solid  
   Always seen in feces, (+) presence in water:
 Semisynthetic agar media (agar based)
 fecally contaminated water
 Middlebrook 7H10 & 7H11  
GENUS UNDER COLIFORM ORGANISMS
 Mitchison’s selective 7H11
  Citrobacter
 Egg potato base medium (Egg based) : routinely used
- Aerobic/ facultatively anaerobic gram (-)
 Lowenstein Jensen Medium Escherichia organism, nonsporogenous, rod-shaped, ferments
o (+) M. tuberculosis Enterobacter lactose to acid w/ gas production
o (-) M. leprae
o Gruft : most routinely used variant Klebsiella - Most impt. Characteristic: GAS PRODUCTION
  ANALYSIS 
STANDARD WATER
o w/ crushed eggs/ egg yolks : heat bet. 35-  
Selective to gram (-) Presumptive test
37C, if > = egg coagulates organism
o malachite green : inhibitor I. Lauryl sulfate (inhibits growth
of gram neg org) lactose broth w/ Durham’s fermentation
o colonies : flat, round, tan in color w. serrated
tube
border (cauliflower colonies)
o Liquid  Pour plate method :
   estimate # of
Broth media
colonies in coliform
 Middlebrooke 7H9 & 7H12 organisms

 Multiple tube technique
(Most Probable Number
method, Multiple tube technique, Dilutional
tube technique)

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BACTERIOLOGY I || Megan Arceño | Cloie Casinillo | Angela Magsucang A.Y. 2015 – 2016, 1st semester

o Principle: replicate liquid broth growth o aerobic/ facultative anaerobic gram negative, non-sporeforming,
in 10-folds dilution rod-shaped, ferments lactose to acid with gas production (most
o Uses 9 lauryl SO4 tubes : # of tubes with important characteristic of coliforms; water analysis (+)
gas formation (only accepted positive o 4 genus: 1) E.coli, 2) Citrobacter, 3) Klebsiella, 4) Enterobacter
result) is compared to MPN table - fecal contamination (the ONLY WAY that the water sample gets contaminated)
o Unit : per 100 mL water   
microorganism in the feces contaminates water sample
  
Confirmed test - NO direct laboratory testing will identify the presence of these bacterial
I.Brilliant green lactose bile broth w/ Durham’s ferm. Tube pathogens in the water sample
    
 Inhibits gram (-) orgs other than coliforms organism not detected in the water (Salmonella typhi, Shigella dysentery)
   
uses another organism that is more measurable/detectable in the sample
(+) result= gas formation only II.
Eosin Methylene Blue agar o indicator of water analysis procedure: E.coli (most abundant
   enteric organism in the GIT of human beings; water is fecally
Purple colonies w/ greenish metallic sheen (+)
 
Large pink colonies (-)
 contaminated
 
Purple colonies w/… and Large pink colonies (+)
 Standard Water Analysis
III.Colilert medium 1) Presumptive Test
    
 content : 4-methylumbelliferone glucoronide (MUG agar) (+) result; presumed organism is present
 
  needs confirmatory test

Chromogenic substrate test : tests ability of E.coli to destroy MUG 
Lauryl sulfate lactose broth with durham’s fermentation tube

content in colilert medium
 All E. coli produce beta glucoronidase that o Lauryl sulfate (detergent ; inhibits the growth of gram negative
organisms)
 destroys gluconoride
  In UV light :
w/ E.coli = fluorescence, MUG destroyed w/o Lauryl SO4 & pour plate method always in
E.coli = no fluorescence, MUG present pair (selective medium)
  
Completed test
I.Nutrient agar slant o evidence of growth in durham’s fermentation tube:
  gas (bubbles)
 Gram staining (gram negative coccobacilli)  
 Pour plate method
II.TSI
 o cannot count entirely ; only the estimate number of
  Acid slant/ acid butt, gas is produced, H2S is not produced III. coliforms present in the sample
IMViC media  
   Multiple Tube Technique (MPN Method – Most Probable Number Method)
 (+)(+)( - )( - )
  o Most accurate number of coliforms present (values lower than
Reporting : The water is/ is not fecally contaminated.
95% Confidence Region)
o dilutional tube technique
o PRINCIPLE: means of replicate liquid broth growth in ten folds
Exercise No. XVI
dilution
Water Bacteriology / Bacteriological Examination of Water
  9 or 15 lauryl sulfate tubes
 1st set (1-3 tubes): 0.1 mL each, 2nd set (4-6 tubes): 1.0
- does not involve chemical testing
mL each (10 folds higher than the 1st set), 3rd set (7-9
- detects presence or absence of microorganism causing disease
- primary objective: determine the potability of water sample from different sources tubes): 10 mL each (10 folds higher than the 2nd set)
   o number of tubes with gas formation is compared to MPN table
1) non-potable – water is unsafe for consumption ; contaminated
   1st set – 1 positive; 2nd set – 2 positive; 3rd set –
 2) potable – water is safe for consumption ; uncontaminated
- intestinal pathogens (microorganisms causing infection in GIT)  1 positive (1:2:1)
    MPN = 150/100 mL of H2O sample
typhoid fever, bacillary dysentery
- microorganism of interest: E.coli & other coliforms in water sample
 
coliforms

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BACTERIOLOGY I || Megan Arceño | Cloie Casinillo | Angela Magsucang A.Y. 2015 – 2016, 1st semester

2) Confirmed Test PURPOSE:

  
presumptive test is positive To determine the sanitary quality of milk by performing a coliform analysis and
 methylene blue reductase test.
 Brilliant Green lactose bile broth with durham’s fermentation  tube (BGLB) o
brilliant green inhibits the growth of other gram negative
organisms other than coliforms MATERIALS:
   1. Unpasteurized milk sample
EMB
  2. Violet Red Bile Agar (VRBA)
Colilert Medium
o 4-methylumbelliferone glucuronide (MUG agar) 3. Sterile NSS for dilution of milk sample
- resuspend positive lauryl sulfate broth
 4. Water bath
- 3 loopfuls inoculate in BG incubate (+) gas
   5. 1:25,0000 Methylene blue (5 drops)
Confirmatory test
- 3 loopfuls EMB (4Q streak plate method)
   PROCEDURE:
Greenish metallic sheen (+) E.coli
 
Large pink colonies (-)
 A. Coliform Analysis
- 3 loopfuls Colilert (4Q streak plate method) 1. Shake the milk sample 25 time. Make dilutions of the unpasteurized milk sample.
   2. Pipette 1 mL milk aliquots of each dilution into an empty sterile petri dish.
Chromogenic substrate test (+)
o tests the ability of E.coli to destroy the MUG content in the 3. Melt and cool the VRBA and add 15 mL of it to each plate. Swirl gently and allow to
colilert medium solidify. Invert and incubate the plates at 35 C for 24 hours.
o all E.coli produces B-glucuronidase, an enzyme that destroys 4. After 24 hours, select the plate that has between 25 to 250 colonies.
glucoronide 5. Count the lens shaped, deep red colonies surrounded by a pink halo. Record this as
 the coliform count per mL of milk (CFU/mL)
o UV light passes through the plate (MUG was destroyed)
fluorescence image (+)
3) Completed Test B. Methylene Blue Reductase Test
   1. Using a sterile pipette, transfer 10 mL of unpasteurized milk to a
gram staining and biochemical testing
   sterile test tube.
nutrient agar slant
   2. Add 1 drop of 1:25,000 methylene blue solution to the tube. Suck
TSI (acid/acid, with gas, no H2S)

IMVIC media (++--)
 and blow several times using the pipette and place a cotton plug.
- Result: The water is fecally contaminated (non-potable) 3. Incubate the tubes in a 37 C water bath and mix again.
4. Observe the tubes at a 30-minute interval for 8 hours. Reduction is
observed by a change of color of the milk sample from blue to
white. When at least 4/5 of the tube has
turned white, the end-point of the reduction has been reached, and the time
Exercise No. XVII should be recorded. Record your results and the class of milk in the report form.
Milk Bacteriology
- Sanitary quality of the milk
 
1) Coliform Analysis
- Pasteurization is a means of processing raw milk to assure that it is relatively free of
o Pour plate method (melted violet red bile agar (VRBA)
bacteria and safe for human consumption. It is a heat process gentle enough to
preserve the physical and nutrient properties of the milk, but sufficient to destroy  deep red colonies surrounded by a pink halo
  
the pathogenic microorganisms. 2) Methylene Blue Reductase Test
  o Indicator dye: 1:25,000 methylene blue
62.9°C at 30 minutes & 71.6°C at 15 seconds
 o Principle: oxygen consumption
o Seal cotton plug – most important procedure
- The presence of coliforms in milk is a major indicator of the sanitary quality of milk.
Their presence is determined by performing a coliform plate count. A high count means  maintains anaerobic environment in the tube
that there is the possibility of the presence of disease-causing bacteria.  lesser oxygen, shorter time for the organism to
consume oxygen
o Detects: END POINT REDUCTION TIME
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BACTERIOLOGY I || Megan Arceño | Cloie Casinillo | Angela Magsucang A.Y. 2015 – 2016, 1st semester

 the time required for the organism to reduce Classification Description End-color
 methylene blue (leuco-methylene blue) product

 blue to white (+) coliform is present Class V Bad White
Class IV Poor Deep pink to
whitish pink
Class III Fair Deep mauve
to deep pink
Class II Good Blue to deep
mauve
Class I Excellent blue
o Factors affecting MB Reductase Test:
 Cold milk
  
More oxygen molecules than warm milk
 
 Increased reduction time

  Pouring back and forth


 from transferring one
More oxygen gets inside
container to another
  
Increased reduction time
 Coliform followed by Strep. Lactis
  
Fastest organism in consuming oxygen
 
 Decreased reduction time
 Increased number of WBCs
  
WBCs consume oxygen
 
 Decreased reduction time
 Light to reduction time (inverse relationship)
  Temperature to reduction time (inverse relationship)
 
3) Resazurin Test
o color of the milk produced after incubation time
o “one hour test” or “three hour test”
o Detects: END COLOR PRODUCT

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