DAY 1 ● Selenite F broth: Salmonella, Shigella (fecal
G9: PREPARATION OF TUBED CULTURE MEDIA specimens); turbidity (+)
Clinical significance: Infectious diseases, GI infections, UTI, ● Tetrathionate broth and Gram-negative broth:
septicemia, bacterial meningitis isolate Salmonella and Shigella from fecal spx
METHODS
1. Nutrient Agar: 2.8 g NA powder, 100 mL dH2O, Enriched v. Enrichment media:
heated Enrichment: liquid; for mixed cultures; supplemented with
2 mL butt 3 mL slant 5 mL butt-slant certain substances to promote the growth of desired
Plated media: dispense after sterilization organisms
(autoclave at 121 degrees C, 15 psi, 15 min) Enriched media: cultivate fastidious organisms
2. Nutrient Broth: 1.3 g NB powder Components of nutrient medium: energy source (carbon,
3 mL in 2 test tubes nitrogen, phosphorus, sulfur, hydrogen, copper, water)
Tubed media: dispense before sterilization
(autoclave at 121 degrees C, 15 psi, 15 min) Broth media over solid media: bulk cultures, volume
testing, growth studies, nutrient accessibility
Culture media, aka bacteriologic media: mixture of artificial
nutrients to provide a controlled …
CHO, AA, Minerals, Vitamins, Salts BLOOD
Uses: blood culture and antimicrobial susceptibility testing
Solidification of agar: red algae polysaccharide agar agar (AST); bacteremia, septicemia, and endocarditis
Agarose: >95 deg C, 50 deg C solidification Collection methods: venipuncture; disinfection with alcohol
and iodine
General isolation media: supports growth of most 20–30 mL for adults, 1–5 mL for children (per culture set)
non-fastidious bacteria; gives no growth advantage; pale Timing: before administering antibiotics and febrile
light yellow to amber episodes
Differential media: provides distinct colonial appearances; Media types: thioglycollate broth
Gram-negative bacteria Manual methods: 35–37 deg C, checked daily
● Sulfide Indole Motility (SIM): butt media; diffusion Automated blood culture systems: BACTEC (CO2 or O2),
away from stab line (+) BacT/ALERT (colorimetric)
● Triple Sugar Iron (TSI): ability of bacteria to
ferment 3 sugars (glucose, lactose, sucrose); G8: PREPARATION OF PLATED CULTURE MEDIA
yellow PURPOSE
● Lysine Iron Agar (LIA): decarboxylation of lysine ● Vital in bacteriology
on the butt portion; deamination of lysine in the ● Provides a controlled environment enriched with
slant portion; purple nutrients
● Simmons Citrate Agar (Citrate): green; intense ● Components: CHO, minerals, AA, salts essential
blue (+); ability of bacteria to utilize citrate as its for bacterial proliferation
sole source of carbon ● Accurate preparation ensures the effectiveness
● Indole broth: production of indole from tryptophan of the media in identifying and analyzing
● Methyl red-Voges-Proskauer: Gram (-), microorganisms
lactose-fermenting Enterobacteriaceae;
PREPARATION
Enrichment broth: prohibit the growth of organism while ● Sterilize before dispensing (100 mL solution)
enhancing that of another ● dH2O
● Thioglycollate broth: aerobes vs. anaerobes; ● Heat until boiling
fastidious anaerobic organisms (e.g., ● Autoclave 15 psi, 15 min, 121 deg C
Actinomyces)
CLASSIFICATIONS
● General isolation (Supportive): facilitate ● Gamma hemolysis: none
proliferation of non-fastidious bacteria
○ NUTRIENT AGAR: beef extract MAC
(necessary vitamins, growth factors), ● Pink colonies (+), positive for lactose
peptone (support metabolic activity), fermentation
agar (solidifying agent), water ● E. coli, K. pneumoniae, Enterobacter spp.
(dissolution of nutrients ● Colorless or translucent colonies (Salmonella,
○ TRYPTICASE SOY AGAR: tryptone, Shigella): (-)
soy peptone, NaCl, H2O, agar
● Non-selective medium ESSENTIAL COMPONENTS:
○ SHEEP BLOOD AGAR: 5% ● Carbon source
defibrinated sheep blood or Type O ● Nitrogen
blood, TSA, brain-heart infusion agar, ● Salts and minerals
Columbia agar ● Water
○ CHOCOLATE AGAR PLATE: Heating, ● Buffer
IsoVitaleX (dextrose, cysteine, vitamin
B12, thiamine, ferric nitrate) COMPONENTS OF MEDIUM (GENERAL)
■ RBCs are hemolyzed; NAD is ● Peptone: provides organic nitrogen in the form of
released AA and peptides
■ Growth of Neisseria spp. ● Beef extracts/yeast extract: supplies
● Differential media: distinct colonial morphologies water-soluble vitamins, CHO, salts, nitrogen
May also be selective; fermentation of glucose compounds
○ MAC-CONKEY: lactose, bile salts, ● Agar: solidifying agent
crystal violet, neutral indicator ● Sodium chloride: osmotic balance of medium
○ EOSIN-METHYLENE BLUE: lactose, (prevent osmotic stress)
eosin dye, methylene blue dye; ● dH2O: facilitates nutrient transport (solvent)
inhibitory ● Buffer: adjusts pH (neutral)
● Selective media: inhibits the growth of all bacteria
except those that are sought WOUND ABSCESS
○ HEKTOEN-ENTERIC AGAR: lactose, 1. SPX COLL.
sucrose, sodium thiosulfate, ferric 2. TRANSPORT
ammonium citrarte, bromthymol blue, Deep: anaerobic transport; superficial: aerobic (2
indicator (stool spx for organisms in GI hr storage, 24 hr processing)
tract) 3. PROCESSING
○ Otherss: MacConkey, EMB, Rejection: contamination with surface materials;
Xylose-lysine deoxycholate (XLD) spx collected with saline contain preservatives;
● Antibiotic media: addition of specific antibiotics requisition and label do not match
○ COLISTIN-NALIDIXIC ACID: SBA Gross exam: color, volume, appearance
base, colistin, nalidixic acid (destruction
of DNA) NAD: FV; Hemin: FX (in CAP)
○ MODIFIED THAYER-MARTIN: CAP
base, vancomycin, colistin, nystatin, G1: INOCULATION TECHNIQUES AND ASEPTIC
trimethoprim lactate TECHNIQUES
Inoculation: introducing microbes into media
BAP Aseptic technique: prevents contamination
● Beta hemolysis: clear zone around colonies, Both are essential in microbiological studies and ensure
complete hemolysis (+) reliable results for research and diagnostics
● Alpha hemolysis: partial hemolysis, greenish PATTERNS
zone (+) ● T-streak
● Quadrant streak ● Diagnosis and microbiological studies
● Continuous streak ● Isolation and study
● Radial ● Prevents contamination in industries
● Overlapping
APPLICATION
iNOCULATION ON TUBED MEDIA ● Clinical labs for diagnosis
● Broth: dip and shake ● Ensures sterile environments in research
● Butt: stab ● Essential in pharmaceutical and food pfoduction
● Slant: streak (bottom to top)
● Butt/slant: stab and streak PURPOSES
● Establish growth of targeted microorganisms
ASEPTIC TECHNIQUE ● Obtain accurate experimental results
● Sterilizing inoculating loops and needles ● Maintain safety in lab settings
● Maintaining clean work surfaces ● Prevents sample and environmental
● Proper handling of tubes and plates contamination
OBJECTIVES CLINICAL SIGNIFICANCE
● Cultivate pure microbial cultures ● Proper diagnosis of microbial infections
● Maintain sterility ● Effective treatment decisions
● Study growth patterns and resistance ● Prevent cross-contamination in clinical labs
● Obtain accurate and reliable results and data ● Protect laboratory personnel from accidentally
acquiring the pathogen
RELEVANCE
● Research and clinical work
DAY 2
G3: EFFECTS OF UV LIGHT
UV light damages bacterial DNA Reasons for incorrect results
Forms thymine dimers, disruots DNA replication ● Delay in UV light exposure
Optimal wavelength: ~260 nm ● Resistance to UV light
● S. aureus needs more time to be exposed with
UV in sterilization UV light
● Used in biological safety cabinets, food
processing, and water treatment Effects of UV light exposure to humans
● Reduces microbial load in healthcare and food ● Benefits: Production of Vitamin D
preparation ● Risks: Short-term overexposure causes
● Limitations sunburns; long-term overexposure may cause
○ Limited penetration: only direct premature aging, skin cancer, and blindness
exposure
Clinical signficance Two types of radiation
● Enhances patient safety ● Non-ionizing (UV light)
● Ionizing radiation (Gamma, X, high-energy
UV steps electron beams)
● Overlapping streak (4 streaks), sterile swab
● Dry in upright position for 5 minutes, cover with Control of microbial growth
aluminum foil ● Damages DNA
● Exposure: 2 hours; inverted incubation 18–24 hrs ● Forms thymine and cytosine dimers
Applications of UV ○ Development of ulcer
● Germinal lamps (hospitals, nurseries, OR) ○ Eject hydrogen ions and modify
● Sterilization of vaccines and other medical proteins
products ● Alkaliphiles
Optimal UV dosage depends on: ○ High pH levels
● Type of microorganism ○ Bacillus cereus: foodborne illnesses;
● Intensity of UV light produce alkaline proteases, lipases,
● Exposure time and amylases; produces tough spores
● Suggestion: 30–40 mJ/cm2 (254 nm)
Effects of pH in bacteria
Effects of pH on bacterial growth ● Cellular processes, enzyme activity, stability of
● pH scale: 0–14 macromolecules
● Most clinically relevant bacteria prefer or grow ● Destruction of hydrogen bonds, DNA, proteins,
best at neutral pH etc.
● Acidic environment inhibits baterial growth
● Acidophiles, alkaliphiles General effects of UV
● Neutrophiles: E. coli, Salmonella spp. ● Skin damage, eye damage, immune suppression,
○ Enzymes maintain stability DNA damage
○ Integrity of cell membrane is preserved
● Acidophiles
○ Low pH levels
○ H. pylori: survive in highly acidic
conditions, secrete urease
Comparisons:
- Stains (Carbolfuchsin—acidic/methylene blue—alkaline, India ink/nigrosin—fine colloidal suspension of colored particles)
Other stains: fluorochromes auramine-rhodamine—auramine O (yellow-green), rhodamine (red); AFB appear yellow to orange
AFB method: Ziehl-Neelsen (by heat) or Kinyoun (by detergent, hello Tergitol)
Negative stain: negative din yung charge ng bacteria (opposites attract kineme, eh same charge kaya may repulsion so
mag-aadhere instead yung stain sa surface)
- Structures (AFB: cell wall with mycolic acid and cord factor wax D, expression of Gram-positive behavior; negative: capsules
kaya di dapat magheat fix kasi maddamage, budding, viruses pag TEM)
*AFB: carbolfuchsin, decolorize with acid-alcohol, methylene blue
- Organisms (AFB: Mycobacteria in TB and leprosy/Hansen’s disease, partially AF Nocardia and Rhodococcus,
Cryptosporidium—enterocolitic, Cyclospora, Cystoisospora belli, Saccharomyces; negative: K. pn, C. neoformans in CSF for
cryptococcal meningitis, smallpox, Ebola)
- Advantages of neg. Stain: minimizes distortion of morphology kasi iniiskip heat fixing
