REVIEW NOTES in BACTERIOLOGY # 1 – REVISED MAY 2023

PART 1 - INTRODUCTION

GENERAL • Classified as PROKARYOTE, reproduce thru binary fission

CHARACTERISTICS • Average size ________________________________________________
OF BACTERIA • Smallest ___________________________________________________
• Longest (10 um) ____________________________________________
• Largest pathogenic bacterium ________________________________
• CELL WALL LESS __________________________________________
With __________________in their cell membrane & Pleomorphic
Classified as Gram ________________________________________
• Spore forming organisms
• ____________________________________

BACTERIAL CELL CELL WALL Give shape to the organism; Provides attachment for flagellum
STRUCTURE a.k.a. • Usual site of antibiotic action, basis of gram staining
peptidoglycan layer or • Disaccharides: N-acetyl-glucosamine & N-acetyl-muramic acid
Murein Layer • Gram positive cell wall with THICKER peptidoglycan layer & with teichoic acid
• Gram Negative cell wall with THINNER peptidoglycan layer & no teichoic acid
• Some components responsible for pathogenicity _________________________________________________________________
CAPSULE • Anti-phagocytic, virulence factor
• Responsible for __________________colonies
• NEUFELD QUELLUNG - ____________________

With polysaccharide capsule S. pneumoniae, K. pneumoniae, N. meningitidis

With Polypeptide D-glutamic Acid:
With HYALYRONIC acid capsule
Polyribosyl Ribitol Phosphate
ALGINATE capsule

PILI • Usually found in gram negative organisms

a.k.a. • COMMON PILI ____________________________________________________________________
• SEX PILI – for gene conjugation, transfer of genetic material ____________________________

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 __________________

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 REVIEW NOTES in BACTERIOLOGY # 1 – REVISED MAY 2023

FLAGELLA • Organ for locomotion

• Most cocci are Non-motile, Bacilli and Spiral organisms are usually motile
• In spiral organisms we call it AXIAL FILAMENTS or PERIPLASMIC FLAGELLA
• MOTILITY is best observed at
Methods to detect Motility
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

Tumbling motility Atrichous Absence of flagella

Twitching motility Monotrichous Single flagellum at one end
Gliding/sliding Examples: Campylobacter, Pseudomonas
motility amphitrichous Single flagellum at both ends i.e., Listeria
Darting motility Lophotrichous Tuft of flegalla at one or both ends i.e., H. pylori
Shooting star Peritrichous Organim surrounded with flagella
motility i.e., Enterobacteriaceae except Klebsiella &
Corkscrew motility Shigella

INCLUSION BODIES • Nutrient storage / stored food

• BABES ERNST BODIES-Volutin seen in __________________________________________
• MUCH granules ____________________________
• Halberstaedter Prowazek -glycogen containing seen in ____________________________________

STAGES OF BACTERIAL GROWTH

- adaptation phase, no increase in number but there is MECHANISMS OF GENE TRANSFER
size - Uptake of NAKED DNA (free DNA)
- No cell division yet - Acquiring DNA from Bacteriophage
- Growth rate 0% - Viris that infects bacteria
- Also called EXPONENTIAL phase ________________________________________
- start of cell division, there us increase in number - DNA transfer via sex pillus
- organism becomes susceptible to antibiotic
Plateau - also called
_________________________________________ - - a miniature chromosome
- characterize by a balance between the number of living - Can be a virulence factor
& dead cells, (+) sporulation - Confers resistance fo antibiotics
- Death Phase/ Phase of Decline

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 REVIEW NOTES in BACTERIOLOGY # 1 – REVISED MAY 2023

BIOSAFETY
LEVELS
BSL 1 Those that can pose _________________ threat Bacillus subtilis
No known potential of infecting healthy people M. gordonae

Precautions: PPE + sterile techniques

Practice standard laboratory techniques
BSL 2 Those that can pose _________________threath Salmonella
Acquired thru ingestion, percutaneous & mucous membrane exposure Shigella
Associated with laboratory acquired infections HBV
HIV
Precautions:
PPE, sterile techniques, contact precautions, vaccination
Use of Biohazard signs, decontaminate all infectious wastes
BSL 3 Those that may pose ________________risk M. tuberculosis
Organisms possible for aerosol transmission
Agents of systemic
Precautions: mycoses
PPE, Sterile technique, aerosol precautions, vaccination
Laboratory must have sustainable airflow in which air is drawn from clean area of the lab towards the contaminated area Coxiella burnetii
BSL 4 Those that may pose _________________risk
Those that can cause life threatening diseases

Precautions:
Class III BSC, maximum containment, Personnel and all materials must be decontaminated before leaving the facility
Laboratory must be situated in a different building

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
• BSL 1 _________________________
CLASS II Also known as _________________________
• BSL 2 & BSL 3 _________________
Sterilizes the AIR ENTERING & CIRUCLATING within THE CABINET & the
• BSL 4 _________________________
EXHAUST AIR
Sterilizes air to be exhausted and the air that flows over infectious material
Additional info:
Class IIA – 70% of air is recirculated Class II B2 Does NOT ALLOW recirculation of
Class IIB – exhaust air is discharged outside the building air
Class II B1 – 30% of air is recirculated a.k.a total exhaust cabinet
CLASS III Class II Routinely used
System is ___________________________ Class IIB For carcinogens
Infectious material is handled using gloves attached & sealed to the cabinet

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 REVIEW NOTES in BACTERIOLOGY # 1 – REVISED MAY 2023

AGENTS OF Easily transmitted & can cause high mortality rate

BIOTERRORISM i.e. B. anthracis, C. botulinum, Y. pestis, F. tularensis
Not as easily transmitted as category A, results in moderate morbidity & mortality rates
i.e Brucella, E. coli 0157:H7, Alpha viruses,
Emerging pathogens that can be engineered for mass spread
Potential for high morbidity & mortality rates
i.e., Hanta and Nipah Viruses

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

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 REVIEW NOTES in BACTERIOLOGY # 1 – REVISED MAY 2023

Depth filter
Cellulose acetate or cellulose nitrate Membrane = liquid 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 – sporicidal, can kill spores in 3-10 hrs, does not corrode lenses, metals and rubbers. use for bronchoscope
• Peracetic acid – surface sterilization of surgical instruments, cold sterilization, effective in the presence of organic material
• Formaldehyde vapor and vapor phase hydrogen peroxide – used to sterilize HEPA filters in BSCs

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
ENDOSPORES SURVIVES in the METHOD

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 spil ls
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
• Phenolics (phenol, Lysol, carbolic acid, hexachlorophene0 – disinfects at high concentration; used in soaps at low concentration
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

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 REVIEW NOTES in BACTERIOLOGY # 1 – REVISED MAY 2023

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- most common cause of sepsis
• 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
• Collect 2-3 sets in 24 hrs, at least 1 hour apart from left & right arms
In adults ≥ 20 ml., in pediatric patients 1-20 ml/set and 1-5 ml in infants and small children BUT 40 ml at one collection in
EMERGENCY CASES where antibiotic therapy must be instituted immediately
• If patient is on antimicrobial, use THIOL BROTH to neutralize antimicrobials or ARD to remove them prior to culture
i.e., ________________________________________________________________-
• Dilution of blood to medium ____________________ Media: TSB, BHI, Brucella broth
• Anticoagulant is .025% Sodium Polyanethol sulfonate (SPS) , this is inhibitory to Neisseria, Gardnerella, P. anaerobius but
to counteract this effect we use ___________________
• White top tube with EDTA – for blood specimens for PCR
• 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 _____________________________________________
• We do rapid testing for cryptococcal antigen
• Usual isolates are ____________________________________________________--
URINE • Specimen of choice for bacterial culture _______________________________________________________________
• Catheterized for those unable to void; SUPRAPUBIC URINE _____________________________________________
• Specimen for molecular studies like PCR __________________________________-----
• Usual request is ________________________________ to rule out UTI
• # 1 cause f UTI _________________________________; UTI in young female _________________________________ and in
ELDERLY WOMEN WITH CATHETER
Other causes of UTI: Klebsiella, E. faecalis
• Perform colony count using calibrated loop to determine # of colonies / ml of urine

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 REVIEW NOTES in BACTERIOLOGY # 1 – REVISED MAY 2023

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 • Patient must be instructed to rinse/gargle with water prior to collection and cough deeply into container
• Processed to diagnose LRTIs like pneumonia, 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: to remove contaminants and normal flora
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 ___ ___
• 1 sputum specimen collected in 3 days, considered (+) __________________________
• 2 specimens in 1 day, considered (+) ________________________
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 ____________________________________
• 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
• If bacterial infection is suspected we collect 3 specimens for 3 days, parasites 3 specimens for 10 days
ADDITIONAL INFORMATIONS
• Usually, specimens are transported to the laboratory _____________ff collection at RT
• Transport of prostatic fluid in glass tubes, suprapubic aspirates must be IMMEDIATE
• CSF, amniotic fluid, synovial, pericardial and all other STERILE BODY FLUIDS except blood must be transported _________________
• Acceptable anticoagulants for PCR ____________________________________________
• For Gastric aspirate specimens, collect early in the morning & before a meal, specimen must be neutralized with SODIUM BICARB ONATE within 1 hr
• For Molecular techniques DO NOT USE HEPARINIZED PLASMA (inhibits polymerase enzyme), Use of PLASTIC SWAB is RECOMMENDED for collection of bacteria, viruses &
Mycoplasma for mucosal membranes, NOT TO BE USED ___________________________________with aluminum shafts (interferes with amp lification of nucleic acids)

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 REVIEW NOTES in BACTERIOLOGY # 1 – REVISED MAY 2023

First voided urine for detection of STIs

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

(+) control – S. aureus Primary Stain/ Initial stain
(-) control – E. coli
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
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)

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 REVIEW NOTES in BACTERIOLOGY # 1 – REVISED MAY 2023

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:
Not agar based
Rice Medium

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 REVIEW NOTES in BACTERIOLOGY # 1 – REVISED MAY 2023

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 _____________

Amie’s _________________, Transgrow ____________
Stuart’s _________________
Todd Hewitt & Lim Broth (Modified Todd Hewitt) ______________________

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

Media for susceptibility test

Motility Test Medium

ADDITIONAL INFORMATIONS
• tap water agar – to differentiate AEROBIC ACTINOMYCETES (Nocardia, Rhodococcus, Tsukamurella, Streptomyces, Actinomadura, Gordonia)
• Plates for SEMIQUANTITATION – streak in 4 quadrants – this is called ISOLATION STREAKING – involves successive dilution of organism until you have the cells at low density
Flame inoculating loop between streaks, to avoid over inoculation
Heavy Growth / many = 4+ / Moderate growth = 3 + / Few or light growth = 2+ / Rare = 1+
• Most common method to MEASURE GROWTH is PLATE COUNT – measures the number of viable cells in milk, food, water and soil (CFU/mL)
ANTIMICROBIAL Purpose: to determine Resistance / susceptibility of an organism to an antimicrobial agent
SUSCEPTIBILITY Involves use of antimicrobials:
TESTING
BACTERICIDAL

BACTERIOSTATIC
With limited range of action
Can act against a number of organisms i.e Tetracycline, chloramphenicol

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 REVIEW NOTES in BACTERIOLOGY # 1 – REVISED MAY 2023

CLASS OF TARGET SITES EXAMPLES IMPORTANT NOTES

ANTIBIOTIC
Beta Lactam Penicillin, Cephalosporins, carbapenems,
monobactam

Glycopeptide Teicoplanin, ________________________

Can also inhibit cell wall synthesis are: ________________________________ and ____________________________________
Aminoglycosides Gentamycin, Tobranycin, kanamycin, amikacin,
streptomycin
Tetracycline Doxycycline, minocycline,
Macrolides- Erythromycin, Clarithromycin
Lincosamide-
Streptogramin
Chloramphenicol Chloramphenicol
Fluoroquinolones Ciprofloxacin, ofloxacin, levofloxacin
Rifampicin
Sulfonamide Folic acid synthesis SXT
Polymyxin Cell membrane Polymyxin, colistin
function
Nitrofurantoin Bacterial enzyme Nitrofurantoin

METHODS OF AST
1. DILUTION METHOD
➢ ________________________________________________________________________________________________________________
➢ This method determines ___________________________________lowest concentration of antibiotic that inhibits bacterial growth
It also determines _______________________________________lowest concentration of antibiotic that kills at least 99.9% of bacteria
➢ Methods:
1) Broth dilution ____________________________________________________________________________________________
Standard inoculum
2) Agar dilution _____________________________________________________________________________________________
Standard Inoculum _______________________________________________________________________________________
Reference method for _____________________________________________________________________________________

2. DISK DIFFUSION METHOD – Kirby Bauer Technique

REQUIREMENTS IMPORTANT NOTES
Thymidine – minimal or absent
MEDIA Increased thymidine = ____________________ to sulfonamides and trimethoprhim
Mueller Hinton Agar / MHA
Calcium (25 mg/L) and Magnesium (12.5 mg/L)
Increased Calcium & Magnesium = decreased activity of aminoglycosides to _____
________________________ and decreased activity of Tetracycline against all
organisms

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 REVIEW NOTES in BACTERIOLOGY # 1 – REVISED MAY 2023

pH: Low pH –
➢ decreased activity of aminoglycosides, erythromycin and clindamycin
➢ increased activity of tetracycline
High pH – decreased activity of tetracycline
Depth of Agar: Too thin agar ______________________________________
Too thick agar _____________________________________
Very dry agar surface _______________________________________
Too much moisture on agar surface ____________________________
INOCULUM subculture 4-5 colonies on TSB Purpose of McFarland _________________________________________________
incubate at 37 degC 3-5 hrs
and compare turbidity of Composition of McFarland ______________________________________________
subculture with 0.5 McFarland Inoculum size” 1.5 x 108 CFU/ml
standard
Inoculum too heavy ____________________________________
Inoculum too light _____________________________________

➢ manner of streaking __________________________________________________________________________

after streaking, wait for _______________________minutes to allow diffusion of organism to entire media
use of MIXED CULTURE can be a source of error

Antibiotic
Disk ➢ Standard size of antibiotic disk ____________________________________
➢ Storage of antibiotic disk: working supply __________________________
Long term storage ________________________________________________________________________
PLATE SIZE
➢ if plate size is 150 mm place no more than __________disks
& NUMBER
OF DISKS ➢ if plate size is 100 mm place no more than __________ disks
DISTANCE ➢ distance of disk from center is ______________________________,
OF DISKS ➢ distance between 2 disks __________________________________

Incubation ➢ invert plates and incubate 35 – 37 degC; for MRSA incubate at ___________________
➢ incubation time ___________________; Aerobic incubation no CO 2 / humidified ambient air
Prolonged incubation ______________________________________

Measuring ➢ Measure zone of inhibition using RULER or CALIPER and interpret if S, R or intermediate
zone of ➢ For media with blood – measure from the top with cover removed
inhibition ➢ If there is SWARMING _______________________________________
➢ When using SULFONAMIDES if there are 2 concentric zones measure _______________________________
Other ➢ Improper storage of disk
Sources of Indicators of Improper storage ___________________________________________
Errors ➢ Reading and clerical errors
➢ Deterioration of turbidity standards or control strains

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When ➢ Use of MHA with 5% Sheep’s RBC is for _________________________________

dealing with ➢ For Mycobacterium we can use ________________________________________
FASTIDIOUS ➢ For MRSA use MHA with 2% NaCl incubated for 24 hrs at 30-35 degC
organisms
➢ N. gonorrhoeae – GC agar with supplements
➢ N. meningitidis use ____________________________________________
➢ For H. influenzae – use of HTM, incubate 35 degC with 5-7% CO2 24 hrs, incubate anaerobically up to 48 hrs
➢ For Anaerobes, use Brucella blood agar with hemin

AUTOMATED SYSTEMS
Vitek System Growth based technology, uses reagent cards and chromogenic substrates
Measures changes in absorbance
Walk away system Fully automated, computer controlled
Bacterial growth may be detected spectrophotometrically or fluorometrically
Phoenix System Involves fluorometric and colorimetric detection, fully automated

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 __________________
• 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
MODIFIED HODGE • Screening test for _____________________________________
TEST • (+) RESULT: clover leaf like pattern of zone of inhibition
ADDITIONAL FREQUENCY OF EQUIPMENT MONITORING
INFORMATIONS Incubator, water bath, refrigerator,
Examples of Critical Values in Microbiology
• Positive blood culture
Freezer, heating block
• Positive cerebrospinal fluid Gram stain or culture
Autoclave – efficiency/spore testing • Positive cryptococcal antigen test or culture
Autoclave temperature • Positive blood smear for malaria
GASPAK jar • Streptococcus pyogenes from a sterile site
Centrifuge function/ rpm • Positive acid-fast smears or positive Mycobacterium culture
Microscopes • Streptococcus agalactiae or herpes simplex virus from
Weighing balance genital site of a pregnant woman at term

Oxidase, catalase, gram stain • Detection of significant pathogen (i.e., Bordetella pertussis, Brucella, Legionella

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 REVIEW NOTES in BACTERIOLOGY # 1 – REVISED MAY 2023

RULE with regards to CRITICAL / PANIC VALUES: ________________________________________________________

BACTERIAL IDENTIFICATION
MANUAL
METHOD
SEMI- Analytical Profile Index-API PCR ______________________________________________
AUTOMATED API 20E, API 20A Steps:
1. _________________________________________________
Uses plastic strips and microtubes with
biochemical substrates. 2. Annealing ________________________________________
The biochemical substrates are inoculated 3. _________________________________________________
with pure culture suspension

AUTOMATED VITEK SYSTEM

MALDI-TOF (Matrix Assisted Laser
Desorption Ionization – Time of Light)

ANAEROBIC CULTURE MICROAEROPHILIC

(Gas Pak Jar) (Candle jar)
5% CO2; 10% H2; 85% N2 5% O2; 10% CO2; 85% N2

• 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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 REVIEW NOTES in BACTERIOLOGY # 1 – REVISED MAY 2023

pg. 15 PREPARED BY: MA. CRISTINA SJ LIWANAG (MAM LIGHT)