REVIEW NOTES in BACTERIOLOGY # 1 – REVISED OCTOBER 2022

CPART 1 - INTRODUCTION
GENERAL • Prokaryote,
CHARACTERISTICS • With both RNA & DNA
OF BACTERIA • Usually with CELL WALL except: _________________________________________
• Some can produce spores _______________________________________________
• Average size __________________________; SMALLEST _____________________________largest pathogenic bacterium ____________________
TOXINS Exotoxins • Produced by gram (+) / (-) ____________________________________________________________________________
• PROTEIN in nature; unstable to heating; effect is local/specific
• Examples: _____________________________________________________________________________________________
Endotoxins • Usually produced by gram (-) organisms __________________________________________________________________
• LPS in nature, stable to heating, effect is systemic/generalized
• Example: _____________________________________________________________________________________________
• LIMULUS LYSATE TEST – detects endotoxins in body fluids, uses aqueous extract of blood cells of horse shoe crabs (+)
result is clumping
BACTERIAL CELL CELL WALL • Main component is _______________________________________
STRUCTURE a.k.a. • Give shape to the organism; Provides attachment for flagellum
peptidoglycan layer or • Usual site of antibiotic action, basis of gram staining
Murein Layer • Disaccharides: N-acetyl-glucosamine & N-acetyl-muramic acid

• With Teichoic acid, thicker peptidoglycan layer

• No teichoic acid, thinner peptidoglycan layer, with outer membrane, LPS and periplasmic space
CAPSULE • Function: _______________________________________________
• Responsible for _________________________________colonies
• Capsular swelling test _________________________________________

With POLYSACCHARIDE CAPSULE: S. pneumoniae, K. pneumoniae, N. meningitidis

Bacillus anthracis ____________________________________________________
H. influenzae ________________________________________________________
____________________________________________________________________
____________________________________________________________________
____________________________________________________________________

• Glycocalyx – less organized compared with capsule, slimy layer, polysaccharide containing i.e., S. mutans
PILI • Usually found in gram negative organisms
a.k.a. • COMMON PILI __________________________________________________
• SEX PILI – for gene conjugation, transfer of genetic material

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 REVIEW NOTES in BACTERIOLOGY # 1 – REVISED OCTOBER 2022

ENDOSPORES • Target of sterilization, contains DIPICOLINIC ACID/calcium dipicolinate

or SPORES • Enables organism to withstand injurious conditions

WITH TERMINAL SWOLLEN SPORES ____________________________________

In B. anthracis spores are ________________ while in C. botulinum __________________

FLAGELLA • Organ for locomotion

• Most cocci are Non-motile, Bacilli and Spiral organisms are usually motile
In Spiral organisms it’s
called Methods to detect Motility
AXIAL FILAMENTS or 1) HANGING DROP
_______________ 2) Use of Flagellar stains: Grays, Leifson
_______________ 3) Use of SEMI-SOLID MEDIA ___________________________________
________________________if growth is AT THE LINE OF INOCULATION
________________________if growth is OUTSIDE THE LINE of INOCULATION
• Motility is best observed at __________________________

Tumbling motility
Twitching motility
Gliding/sliding
motility
Darting motility
Shooting star
motility
Corkscrew motility
BACTERIAL CELL INCLUSION BODIES
STRUCTURES • Metachromatic granules-BABES ERNST BODIES-Volutin seen in _________________________________________
• MUCH granules __________________________________________
• Halberstaedter Prowazek -glycogen containing seen in ______________________.
Can be demonstrated using Gimenez, Macchiavelo, and Castaneda
Other structures • CELL MEMBRANE – phospholipid bilayer, No STEROLS except in _______________________________
• NUCLEOID – contains the genome
• RIBOSOME – mediates protein synthesis

STAGES OF • ____________________- adaptation phase, no increase in number but there is __________________

BACTERIAL No cell division yet
GROWTH • ______________ or EXPONENTIAL phase – start of cell division; growth rate starts to increase, organism becomes susceptible to antibiotics
• Plateau or ________________________- characterize by a balance between the number of living & dead cells, (+) sporulation
• Death Phase/ Phase of Decline

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 REVIEW NOTES in BACTERIOLOGY # 1 – REVISED OCTOBER 2022

BIOSAFETY
LEVELS

BIOLOGICAL
SAFETY CABINET CLASS 1 Negative pressure, ventilated cabinet. Unsterilized air enters and circulates within the cabinet, & the exhaust air from the cabinet is
filtered by HEPA filter
Sterilizes only the air to be exhausted
CLASS II Also known as _________________________
Sterilizes the AIR ENTERING & CIRUCLATING within THE CABINET & the EXHAUST AIR
Sterilizes air to be exhausted and the air that flows over infectious material

Class IIA – 70% of air is recirculated

Class IIB – exhaust air is discharged outside the building
CLASS III
System is ___________________________
Infectious material is handled using gloves attached & sealed to the cabinet

• BSL 1 _________________________
• BSL 2 & BSL 3 _________________
• BSL 4 _________________________

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 REVIEW NOTES in BACTERIOLOGY # 1 – REVISED OCTOBER 2022

METHODS OF Destruction & removal of all forms of microbial life including their spores (Physical or Chemical)
MICROBIAL
Destruction & removal of pathogens but not necessarily all microorganisms & their spores (Physical or Chemical)
CONTROL

PHYSICAL METHODS OF STERILIZATION

USE OF MOIST HEAT AUTOCLAVING - Principle ____________________________________________________
Autoclaving • 121 degC 15 psi 15 mins _____________________________________________
Tyndallization • 121 degC 15 psi 30 mins – to sterilize contaminated biological materials
Inspissation • 132 degC 15 psi 30-60 mins – to sterilize infectious medical wastes
• Biological indicator: Bacillus stearothermophilus
• Not killed by autoclaving ______________________- infectious protein particles which may cause
neurological diseases in animals & man i.e., Mad Cow Disease, Creutzfeldt Jacob Syndrome,
Bovine spongiform encephalopathy

FRACTIONAL / INTERMITTENT STERILIZATION _____________________________________

• _______________________________ carried out at 100 degC for 30 mins for 3 days using Arnold
sterilizer, uses flowing steam
• _______________________________ For sterilizing media with increased protein like Lowenstein
Jensen; carried out at 75 – 80 degC for 2 hrs for 3 consecutive days, thickening thru evaporation
• With FRACTIONAL STERILIZATION method, killed
On the 1st day _______________________________
On the 2nd day _______________________________
On the 3rd day _______________________________

USE OF DRY HEAT INCINERATION OVEN CREMATION FLAMING

Use of Oven 870-980 degC 160-180 degC
Flaming Can eliminate prions 1-2 hrs
Incineration Biological Indicator:
Cremation Bacillus subtilis var. niger
FILTRATION Membrane Filter
Millipore filter – with pore diameter of 0.22 u can give 100% sterility
HEPA filter – can remove objects larger than 0.3 u
Depth filter
IONIZING RADIATION Use of Gamma Rays
Biological indicator: Bacillus pumilus
CHEMICAL METHODS
• Chemical sterilants are also called BIOCIDES
• Most commonly used chemical sterilant & is used for machines that cannot be autoclaved________________________________
Biological indicator: Bacillus subtilis var globijii
• Use of glutaraldehyde & peracetic acid is also called ________________________________________
• Formaldehyde vapor and vapor phase hydrogen peroxide – used to sterilize HEPA filters in BSCs

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 REVIEW NOTES in BACTERIOLOGY # 1 – REVISED OCTOBER 2022

PHYSICAL METHODS OF DISINFECTION

Non-Ionizing Radiation UV exposure
Boiling 100 degC 15 – 30 minutes
Pasteurization 63 degC 30 mins (Low temperature Holding) – known as Batch Method or VAT Pasteurization
72 degC 15 secs – HTST/High temperature short time or Flash method
140 degC 3 sec – Ultra High Temperature short time

CHEMICAL DISINFECTION: Antiseptics – for use on the skin while Disinfectants – for use on surfaces
ANTISEPTICS:
• Most commonly used _______________________
• Forms of Iodine 1. Iodine + alcohol __________________________ 2. Iodine + detergent ________________________
• Best antiseptic is _________________________
• 10& hydrogen peroxide – for cleansing of wounds
• 70% ethanol is more effective than 95% EA as disinfectant
• Ethyl alcohol & isopropyl alcohol are non-SPORICIDAL
DISINFECTANTS:
• Best disinfectant for Blood spillage: sodium hypochlorite; CDC recommends ____________dilution of bleach to remove blood spills
For porous surfaces ________________________ while for smooth & hard surfaces ____________________________
Contact time for HBV inactivation ____________________while for HIV inactivation ______________________
• STANDARD: Phenol
• Can be used as substitute for household bleach ________________________________________
• QUATS
SPECIMENS, Important Rules • When to collect specimen _____________________________________________________________________________
SPECIMEN • Observe _________________________, collection containers must be properly labeled & must be sterile
COLLECTION & • Adequate amount must be collected.
HANDLING • Collected samples must be transported without delay ______________________________________________
• In the processing of samples, observe LEVEL of PRIORITIZATION
PRIORITIZATION of SPECIMENS
1 Critical / Invasive CSF, Amniotic fluid, blood, pericardial fluid, heart valves
2 UNPRESERVED feces, sputum, wound drainage
3 Quantitation required Catheter tip, urine, tissues for quantitation
4 Preserved urine, feces, swab in holding media
5
BLOOD • Purpose of blood culture _____________________________________________________________________________
Blood pathogens: E. coli, P. aeruginosa, S. aureus
• __________________________________________________________________________________________
• Phlebotomy site must be cleansed with 70-95% alcohol- Iodine scrub- alcohol rinse; chlorhexidine maybe used instead of
iodine
• Common contaminants: P. acnes, S. epidermidis, diptheroids, Viridans Strep

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 REVIEW NOTES in BACTERIOLOGY # 1 – REVISED OCTOBER 2022

• Dilution of blood to medium ____________________ Media: TSB, BHI, Brucella broth

• Anticoagulant ______________________________________________________________________________________
___________________________________________________________________________________________________
• Not to be used for bacteriologic culture ________________________________________________________________
• Anticoagulant for viral culture but inhibitory to gram + organisms & yeast ___________________________________
• Routine blood culture bottles are held for 7 days observe for signs of growth : turbidity, hemolysis, pellicle formation
For Brucellosis detection ________________________for leptospirosis detection ______________________________
CSF • Collect in 3 tubes, use tube ______________ for micro; collection is thru lumbar tap
• Process ASAP; Storage temp ______________________ transport temp _____________________________
• For smear preparation & culture use _____________________________________________
URINE • Specimen of choice for bacterial culture _______________________________________________________________
• Catheterized for those unable to void; SUPRAPUBIC URINE _____________________________________________
• Usual request is ________________________________ to rule out UTI
• # 1 cause f UTI _________________________________; UTI in young female _________________________________
Other causes of UTI: Klebsiella, E. faecalis
• Perform colony count using ____________________________loop to determine # of colonies / ml of urine
Considered UTI is colony count of ______________________________________________
Formula:
# of colonies counted x dilution factor = colony count/ml of urine
Dilution factor if 1 ul loop was used ________________________if 10 ul loop was used ________________________
SPUTUM • Evaluate quality, Do Bartlett’s Classification ____________________________________
If more than 10 SECs and less than 25 PMNs were noted ___________________________
• For M. tuberculosis identification, we do ________________________________________________________________
Purpose of decontamination __________________________________________________________________________
Purpose of Digestion ________________________________________________________________________________
Gold standard for digestion & decontamination _________________________________________________________
Other agents:
✓ Z-TSP (zephiran & Trisodium phosphate)
✓ 4% NaOH
✓ Cetylpyridium chloride Sodium chloride method
✓ 5% oxalic acid ______________________________________________________________________________
• M. tuberculosis is classified as a BSL ___________ pathogen and therefore culture must be done using BSC Class
____________
THROAT SWAB • Identification of ________________________________which is considered as the major throat pathogen
NASOPHARGYNGEAL • Normal throat flora _________________________________________________________________________
SWAB • Nasopharyngeal swab may be required to detect carrier state of ______________________________ and identification of B.
pertussis & H. influenzae
• For bacterial culture use Dacron, Calcium alginate. Toxic to Neisseria ____________________________________

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 REVIEW NOTES in BACTERIOLOGY # 1 – REVISED OCTOBER 2022

• For Viral culture, Dacron, Cotton, Rayon fibers. Toxic to viruses _________________________________________
STOOL • Detection of GIT pathogens like ________________________________________________
• Maybe collected if stool collection is not possible _________________________________
• # Of quadrants streaked on plated media ________________________________________
• Gram staining is not usually done
STAINING OF Use of only 1 dye, the color of the dye is the resulting color i.e. methylene blue
BACTERIAL CELLS Only the background and not the organism is stained i.e. india ink
Organism appears colorless
SPECIAL STAINING Use to demonstrate special features of the cell
Capsular stains Hiss, Anthony’s, Tyler, Muir
Spore stain Dorner’s, Schaeffer and Fulton, Wirtz and Conklin
Flagella Grays, Fisher and Conn, Leifson
Metachromatic granules Albert’s, Neisser, Ljubinsky, Ponder,methylene Blue, Lindergran, Burke’s technique
Polar Bodies Wayson stain,
Methylene Blue
spirochetes Levaditi’s
DIFFERENTIAL To differentiate one organism from another
STAINING Examples:

GRAM STAINING PURPOSE Reagent Gram Positive Gram Negative

Primary Stain/ Initial stain

Mordant

Decolorizer

Counterstain
Secondary stain

GRAM POSITIVE: _________________________________GRAM NEGATIVE: __________________________________

Most Critical step in gram staining ________________________________________________________

Hucker’s modification __________________________________________________________________
Carbol fuchsin can be used as counterstain in place of safranin. This may be done to improve staining of some gram-negative
organisms that are poorly stained with safranin

Rules:
1. All COCCI are gram (+) except Neisseria, Branhamella and Veilonella
2. All BACILLI are gram (-) except:
• Mycobacterium, Corynebacterium, Clostridium, Bacillus, Erysipelothrix, Lactobacillus, Listeria

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 REVIEW NOTES in BACTERIOLOGY # 1 – REVISED OCTOBER 2022

3. Higher forms of organisms like Actinomyces, Nocardia, Streptomyces, yeast and molds are gram (+)
4. All Spiral organisms are reported as Gram (-)
• Not GRAM stained are
Rickettsia; Chlamydia (intracellular); Mycoplasma; Ureaplasma (wall less); spirochetes (can’t resolved by bright field)
ACID FAST STAINING Purpose Ziehl Neelsen / Hot method Kinyoun’s / Cold method Auramine-Rhodamine
(Fluorochrome)
Primary
Mordant
Decolorizer
Counterstain
Result Acid Fast: _________________________________
Non-Acid fast: ______________________________
May be used as substitute for
Methylene Blue Counterstain

ACID FAST ORGANISMS – are organisms that are very hard to stain but once stained they are difficult to decolorize due to MYCOLIC
ACID / HYDROXYMETHOXY ACID that envelopes the bacteria
Rule: All bacteria are Non-Acid Fast except: MYCOBACTERIUM, slightly acid fast is NOCARDIA
Other Methods of Acid-Fast Staining:

✓ Pappenheim’s (urine) to differentiate M. smegmatis _________ from M. tuberculosis _________________

✓ Baumgartens (tissue) to differentate M. leprae _____________ from M. tuberculosis _____________
✓ Fite Faraco’s M. leprae ________________________
CULTURE & Any medium that contains all what is needed to support bacterial growth
CULTURE MEDIA • AGAR usually derived from red algae, solidifies at ________________ & melts at _________________
• Cooling temperature for distribution of media to plate ___________________________________________
• amount: ____________________________________
Growth of organism in culture i.e Pure culture, mixed culture, stock culture

Types as to consistency LIQUID SEMI-SOLID SOLID BIPHASIC

Solidifying agent/ agar 0% 0.5-1% 2-3%

Examples TSB SIM Liquefiable: HBT

Alkaline EMB
Peptone MSA
Water
BHI Non-liquefiable:
Rice Medium

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 REVIEW NOTES in BACTERIOLOGY # 1 – REVISED OCTOBER 2022

Types as to purpose
General Purpose Contains only what is needed to support bacterial growth

Enrichment media To enhance bacterial growth

Enriched media For growing fastidious organisms like

Contains blood
Selective media Used to promote growth of a particular organism & at the same time preventing
growth of other

It contains inhibitor

Differential Media Used to differentiate organisms that are growing together

Selective & Differential Examples:

Transport Media Purpose:

JEMBEC, Cary Blair, Transgrow, Amies

Biochemical test media Used for biochemical testing which aids in bacterial identification

Media for susceptibility test

Motility Test Medium

ANTIMICROBIAL Methods 1) Dilution Method

SUSCEPTIBILITY 2) Disk Diffusion Method
TESTING KIRBY BAUER DISK DIFFUSION METHOD
MEDIA Mueller Hinton Agar: pH ________________ Depth of Agar_________________ Uses filter paper disk = 6mm
INOCULUM • subculture 4-5 colonies on TSB incubate at 37 degC 3-5 hrs
• Compare turbidity of subculture with McFarland standard purpose: __________________________________
• 0.5 McFarland Standard ____________________________________________________________________
• Equivalent of McFarland Standard – CFU _______________________________________________________
• If turbidity ok, inoculate on MHA streaking method _____________________________use of sterile cotton swab.
• if too turbid, dilute using NSS or distilled water (overlap streaking)
• Wait for 3-5 minutes before applying the disk

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 REVIEW NOTES in BACTERIOLOGY # 1 – REVISED OCTOBER 2022

PLATE SIZE • if plate size is 150 mm place no more than __________disks, if plate size is 100 mm place no more than ________
NUMBER OF disks
DISKS • distance of disk from center is ______________________________, between 2 disks ___________________
DISTANCE OF • invert plates and incubate 35 – 37 degC ; incubation time ___________________ but for MRSA _______
DISKS ; Aerobic incubation no CO2
• Measure zone of inhibition using RULER or CALIPER and interpret if S, R or intermediate
• For media with blood – measure from the top with cover removed
• For Streptococci, MHA is supplemented with 5% Sheep’s blood
SOURCES OF
ERRORS • Too light inoculum ___________________________ Inoculum too heavy ___________________________
• Depth of agar: Thin ___________________________ Thick ______________________________________
• Used of mixed culture
• Too much moisture on surface of agar __________________Very dry agar ___________________________
• Improper storage of disk Storage temp for antibiotics WORKING SUPPLY ___________________________
Long term storage ____________________
Acid pH affect result in tetracycline, Novobiocin, Methicillin = FALSE SUSCEPTIBLE
Alkaline pH affect result in aminoglycosides/erythromycin = FALSE SUSCEPTIBLE
• Prolonged incubation_________________________________
• If there is SWARMING __________________________________
• When using SULFONAMIDES if there are 2 concentric zones measure _______________________________

ANTIMICROBIAL
AGENTS • Beta Lactams – Penicillin, Carbapenems, Caphalosphorins
• ___________________- vancomycin
PROTEIN SYNTHESIS INHIBITORS • ___________________ - erythromycin
• ___________________ - gentamicin, tobramycin
• ___________________ - doxycycline
• Chloramphenicol
BETA LACTAMASE INHIBITORS Tazobactam, Sulbactam, clavulanic acid

SULFONAMIDES SXT ____________________________________________________________

Drug which can be used as treatment for UTI; inhibits bacterial enzyme
QUINOLONES Inhibition of DNA activity
Examples: Levofloxacin, Ciprofloxacin
MISCELLANEOUS • To detect inducible clindamycin resistance among strains of S. aureus
TESTS • Uses 15 ug Erythromycin & 2 ug Clindamycin which are positioned 15 mm apart
• (+) result: Blunting /flattening of the Clindamycin zone to produce a D pattern
• Interpretation of Result:
(+) D-test- we report it as Clindamycin _________________
(-) D-test- we report it as Clindamycin _________________
• Gene that activates resistance to clindamycin __________________

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 REVIEW NOTES in BACTERIOLOGY # 1 – REVISED OCTOBER 2022

• Uses a strip with single antibiotic of different concentrations along its length
• Dilution method
• Suited for fastidious organisms & anaerobes
Serum Bactericidal • A.k.a. ___________________________________________
Test • Measures activity of antibiotics in patient’s own serum against the pathogen, to detect if patient is
receiving effective treatment for infection
Penicillin Resistance • For ____________________________
• Uses MHA supplemented with sheep’s blood + 1 ug oxacillin disk
• Greater than 20 mm ___________; less than 20 mm ____

MODIFIED HODGE • Screening test for _____________________________________

TEST • (+) RESULT: clover leaf like pattern of zone of inhibition

ADDITIONAL
INFORMATIONS FREQUENCY OF EQUIPMENT MONITORING RULE ________________________________________________________

Incubator, water bath, refrigerator Examples of Critical Values in Microbiology

Freezer, heating block • Positive blood culture
Autoclave – efficiency/spore testing • Positive cerebrospinal fluid Gram stain or culture
Autoclave temperature • Positive cryptococcal antigen test or culture
GASPAK jar
• Positive blood smear for malaria
Centrifuge function/ rpm
• Streptococcus pyogenes from a sterile site
Microscopes
• Positive acid-fast smears or positive Mycobacterium
Weighing balance
culture
Oxidase, catalase, gram stain
• Streptococcus agalactiae or herpes simplex virus from
genital site of a pregnant woman at term
• Detection of significant pathogen (i.e., Bordetella
pertussis, Brucella, Legionella

BACTERIAL IDENTIFICATION
MANUAL METHOD PCR ______________________________________________
SEMI- Analytical Profile Index-API
AUTOMATED API 20E, API 20A Steps:
1. ________________________________________________
Uses plastic strips and microtubes with biochemical
2. Annealing ________________________________________
substrates.

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 REVIEW NOTES in BACTERIOLOGY # 1 – REVISED OCTOBER 2022

The biochemical substrates are inoculated with pure 3. ________________________________________________

culture suspension
ANAEROBIC CULTURE MICROAEROPHILIC
AUTOMATED VITEK SYSTEM (Gas Pak Jar) (Candle jar)
MALDI-TOF (Matrix Assisted Laser Desorption
5% CO2; 10% H2; 85% N2 5% O2; 10% CO2; 85% N2
Ionization – Time of Light)
• GASPAK JAR ______________________________; Uses Palladium catalyst
• Most common failure of GasPak jar _________________________________________________________
• Indicators: Methylene Blue or Resazurin. In the presence of oxygen Methylene Blue is ________ while Resazurin is ______________,
but in the absence of oxygen, they become _________________
• CANDLE JAR _______________________________: Examples __________________________________
• Benchmarking means – peer comparison
• Brightfield microscope is also known as _____________________________________
• Purpose of boiling thioglycolate ____________________________________________

Most effective way to stop the chain of infection

Type of precaution applied to all HUMAN BLOOD & BODY FLUIDS that contain visible blood
STANDARD PRECAUTION To treat patient’s blood & body fluid as POTENTIALLY INFECTIOUS
DONNING Gown-mask-goggles or face shield and gloves
DOFFING Gloves-goggles-gown-mask, then do handwashing after

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