STAINING PROCEDURES:-used to provide proper identification of microorganisms (size, shape, morphology)-requires bacterial smears on glass slides, air-dried, & fixed prior to staining

Methods of fixation:1) Heat fixation (by alcohol lamp, Bunsen burner)2) Methanol fixation 30secs

Purposes of fixation:1) to kill microorganisms2) to preserve the morphology/shape3) to anchor the smear on the slide

Types of Staining:1) Simple staining applying only one stain upon fixation (e.g. methylene blue)2) Structural staining applying special stains to identify special structures (flagella, capsules, endospores)3) Differential staining using sets of stains (primary & secondary stains) to compare groups of microorganisms e.g. as Gram-positive or Gram-negative organisms*proposed by Dr. Hans Christian Gram

Steps in Gram stain:1. Heat-fix slide. Air-dry. Flood with crystal violet (1min)2. Rinse slide. Flood with iodine solution (1min)(note:ALL organisms appear purple)3. Rinse slide. Decolorize with acetone (5secs)4. Wash slide with water. (note: gram-negative organisms turns colorless)5. Apply safranin (counterstain) 30 secs6. Wash with water. Air-dry. Examine under Oil Immersion (OIO)Note: After the last step, Gram-positive organisms appear blue-purple; while Gram-negative organisms appear pink-red structures. Colorless ones are identified as Gram-variable (e.g. Mycobacterium, Nocardia spp)Note: Gram-positive organisms are stained well with crystal violet because of the thicker layer of peptidoglycan (cell wall) compared to thin layers of Gram-negative ones.Note: Gram-negative organisms are stained well by safranin because of the thinner layer of peptidoglycan (cell wall) and presence of lipopolysaccharides enables crystal violet-iodine stain easily washed away once decolorized by acetone.

*Acid-fast stain:-Differential staining which helps identify Mycobacterium spp. From other bacteria-uses carbol-fuchsin (red dye) as the primary stainApplied with heat (to remove the wax of Mycobacterium cell wall that makes the organism red bacilli (from carbol-fuchsin-primary stain) against blue background (from methylene blue- counterstain). Acid-fast organisms are NOT easily decolorized by the acid-alcohol.-usually employed for sputum analysis to detect TB-developed by Paul Ehrlich

Methods of Acid-fast stain:1) Hot method (Ziehl-Neelsen)2) Cold method (Kinyouns)

STUDY p.60 (table 4-6) about staining reaction-morphology-examples of bacteria

MICROBIAL ECOLOGY:-study of interrelationships between microbes and other organisms (living or non-living)-most relationships between humans and microbes are beneficial rather than harmful

*Symbiosis living together of two different organisms (symbionts)Types:1) Mutualism both symbionts benefitse.g. humans provides food to microflora (E.coli) while E. coli provides Vitamin K essential to humans; termites provide shelter to intestinal protozoans while these protozoans helps termites in digestion of wood; LICHENS groups of algal cells provide food for fungi by photosynthesis while groups of fungal cells protect the alga from dessication(drying)2) Commensalism only one symbiont benefitse.g. Demodex mite lives on human hair follicles in order to survive while human is NEITHER benefitted nor harmed3) Parasitism one symbiont is harmede.g. protozoan Trypanosoma gambiense (RBC parasite) causes African sleeping sickness to humans 4) Neutralism neither sides are affected

INDIGENOUS MICROFLORA:-microbes that resides in and on the human body.-variations of the following factors (moisture, pH, nutrients) makes differences of microflora throughout the body

STUDY p.162 (table 10-1) Anatomic locations of bacteria and yeasts microflora

*superinfection overgrowth of microflora as a result of imbalance between other resident microflora. (e.g.Candida albicans microflora causing moniliasis)

Microflora of the SKIN:-most common (Staphylococcus epidermidis, Corynebacterium, Propionibacterium)-mostly anaerobes

Microflora of the EARS & EYES:-middle ear, inner ear usually sterile-outer ear with microflora-sneezing, coughing sometimes causes infection to middle ear-LESS number of microflora on eye surfaces due to normal secretion of tears and lysozymes

Microflora of the RESPIRATORY TRACT:-MORE microflora on the upper resp tract (nose, pharynx) ; more moisture-Sterile on the lower resp tract (larynx, trachea, bronchi, lungs) ; due to phagocytic cells macrophages in the lungs

Microflora of the ORAL CAVITY:-numerous aerobic & anaerobic microflora-most common : Streptococcus spp.-Streptococcus mutans causing dental plaques

Microflora of the GASTROINTESTINAL TRACT:-only Helicobacter pylori survives as microflora of the stomach with very low pH (acidophile)-LESS microflora in the DUODENUM of small intestine because of BILE (inhibits bacterial growth)-MORE microflora in the JEJUNUM & ILEUM of small intestine-LARGE INTESTINE with the largest number of microflora mostly anaerobes

Microflora of the GENITOURINARY TRACT:-normally, the kidneys, ureter & urinary bladder are sterile areas-Escherichia coli from colon causes UTIs when it gains access to the urinary bladder-distal urethra is much prone to infections due to sexual intercourse & poor hygiene-normally, the reproductive systems of both male and female are sterile EXCEPT the external genitalia (vagina & penis)-during puberty and menopause, vaginal secretions are more alkaline (allows growth of E. coli, Strep, Staph)-during childbirth, vaginal secretions are more acidic (allows growth of Lactobacillus acidophilus producing lactic acid which inhibits growth of other bacteria causing vaginosis such as caused by Gardnerella vaginalis)*vaginitis : vaginal infection with presence of PMNs *vaginosis : vaginal infection with NO PMNs

BENEFICIAL ROLES OF MICROFLORA:-provides nutrients (vitamin K & B12, pantothenic acid, pyridoxine, biotin)-provides space within the body to prevent colonization of foreign microbes-constantly stimulates the immune system thereby producing antibodies

MICROBIAL ANTAGONISM involves the following:1) indigenous microflora competes with invading foreign microbes2) production of antibiotics using certain bacteria & fungi to kill other pathogenic microbes

OPPORTUNISTIC PATHOGENS:-certain microflora may gain access to sterile body regions-certain foreign microbes which are usually nonpathogens, becomes pathogenic for IMMUNOCOMPROMISED person

BIOTHERAPEUTIC AGENTS (probiotics)-bacteria and yeasts from EXOGENOUS sources may be ingested to reestablish and stabilize imbalance of microforae.g. Lactobacillus, Bifidobacterium (bacteria)Saccharomyces (yeasts)

BIOFILMS-complex communities of assorted microbes-usually common on indwelling medical devices (prosthetic heart valves, catheters, implants)-provides antibiotic-resistance as some bacteria protects other species of bacteria within the biofilm-bacteria supports each other to promote metabolism-provides resistance to host defense (biofilms weakens phagocytic activity of leukocytes frustrated phagocytosis)

SYNERGISTIC INFECTIONS (polymicrobial/ mixed)-2 or more microbes when combined becomes an effective pathogene.g. ANUG trench mouth/ Vincent disease(Fusobacterium + Treponema + Bacteroides + Prevotella)

AGRICULTURAL MICROBIOLOGY:-microbes provides genetically engineered plants-microbes are used as pesticides-microbes are decomposers (saprophytes)

*NITROGEN CYCLE-nitrogen-fixing bacteria (converts nitrogen gas (N2) from air to ammonia (NH3) and ammonium ion (NH4+) e.g. Rhizobium-nitrifying bacteria (soil bacteria that converts ammonium ions into nitrite (NO2-) and nitrate ions (NO3-) e.g. Nitrobacter, Nitrosococcus-denitrifying bacteria (converts nitrates to artmospheric Nitrogen gas) e.g. Pseudomonas, Bacillus*ammonification involves animal waste (urine) excretion, by certain bacteria converts urea to ammonia

*SOIL MICROBES-usually saprophytes-some are pathogens: with bacterial spores (Clostridium tetanii, Bacillus anthracis); with fungal spores (Cryptococcus neoformans)ANIMAL DISEASES see p168 (table 10-2)

PLANT DISEASES see p169 (table 10-3)

MICROBIAL BIOTECHNOLOGY:-human genes introduced to bacteria and yeasts to produce therapeutic products (human insulin, HepaB vaccine)-production of vitamin B2 (riboflavin); B7 (biotin); B9 (folic acid); B12-production of antibiotics (peniciilins from fungi; bacitracin from bacteria)-production of foods (milk, bread, cheese)-production of alcoholic beverages (beer, wine)-production of chemicals (acetone, ethanol)-bioremediation (use of microbes to clean up industrial wastes) DIAGNOSIS OF INFECTIOUS DISEASES:-depends on patient history, signs and symptoms, and proper collection & processing of clinical specimens

*CLINICAL SPECIMENS blood, urine, stool, CSFSTUDY p221 (table 13-1)STUDY p222 (figure 13-1)

*STANDARD PRECAUTIONS-consider all clinical specimens as potentially infectious-wear personal protective equipments PPEs before, and during specimen collection & processing-all specimens should be collected into a leakproof primary container; also requires a second container

*COMPONENTS OF SPECIMEN QUALITY1) proper selection of specimen2) proper collection of specimen3) proper transport of specimen

BLOOD:*plasma liquid portion of unclotted blood*serum liquid portion of clotted blood*cells WBCs RBCs platelets

STUDY p224 (figure 13-2)55% fluid 45% formed elementsBuffy coat (WBCs & platelets)

-blood collection (PHLEBOTOMY) for blood culture requires ASEPTIC TECHNIQUE (Alcohol-iodine) prior to INSERTION OF NEEDLE-blood culture bottles are promptly sent to the lab for incubation at 37degC (within 5 days)

URINE:-clean-catch, midstream urine is ideal for culture-urine culture includes colony count, identification of pathogen & sensitivity testing-urine culture requires calibrated loop for inoculation of specimene.g. colony count - 100,000 CFU/mL of E. coli

CSF (cerebrospinal fuid): -collection by LUMBAR PUNCTURE-CSF culture is a STAT procedure-preliminary results (gram stain) are relayed immediately

SPUTUM:-pus collected deep down the lungs of a patient with pneumonia, tuberculosis-proper collection requires deep cough-usually stained for Acid-fast (Mycobacterium tuberculosis)-Needle biopsy (FNAB) may be used to recover Pneumocystis jiroveci (AIDS patients)

THROAT SWABS:-used to determine Strep throat (Strep. pyogenes)or others (Neisseria gonorrhoeae, Corynebacterium diphtheriae)

WOUNDS:-better use aspirates than swabs-indicate the type of wound and site of collection

GC Cultures: (gonococci)-to determine Neisseria gonorrhoeae (fastidious, microaerophile, capnophile)-use Dacron swabs inoculated to Thayer-Martin agar

FECES:-used to recover Gram-positive (Clostridium difficile)and Gram-negative (Samonella, Shigella, E. coli) organisms, fungi (Candida), and protozoans (Giardia, Entamoeba)

CLINICAL MICROBIOLOGY LABORATORY:-under CLINICAL PATHOLOGY

*Bacteriology section*-performs routine Gram stain, Acid-fast stain, Culture and Sensitivity testing-performs smear preparation, inoculation, streaking, staining, incubation, microscopic examination-provides environmental culture (to monitor outbreaks or possible NOSOCOMIAL infections)

*Virology section*-use of electron microscopy, molecular assays,and cell cultures (CYTOPATHIC EFFECT)

*Mycology section*-use of KOH smear, Saborauds dextrose agar (SDA), lactophenol cotton blue (LPCB)

*Parasitology section*-use of microscopy to study the characteristic appearance (trophozoites, cyst stage)

*Mycobacteriology section*-TB Lab-performs acid-fast stain, TB culture (LJ medium)