Clinical Bacteriology Laboratory Notes BSMLS-3B2 PDF

Clinical Bacteriology Laboratory Notes I BSMLS-3B2 MODULE 1 BIOSAFETY - mostly caused by viruses - full body, air- OVERVIE...

Clinical Bacteriology Laboratory Notes I BSMLS-3B2 MODULE 1 BIOSAFETY - mostly caused by viruses - full body, air- OVERVIEW OF SAFETY PRACTICES leading to instant death supplied positive LEVEL 4 - agents that pose high risk of life pressure suits 1. All users of the bacteriological lab must focus on the need (BSL-4) threatening diseases for safety. - the highest level of biological 2. There are a lot of hazards that must be acknowledged and safety guarded against. - exposure can be fatal and without treatment or vaccines 3. Users must be aware what hazards exist, the basic safety - requires the use of full body, precautions associated with them, and how to apply air-supplied positive suits remedies in case of accidents (e.g. Ebola virus, small pox, 4. Body contact, specifically hand contact is the primary mode SARS, influenza virus, Marburg virus) of infectious transmission. HAZARD VS RISK *Note: Hand washing is recommended to reduce exposure to Hazard – a potential to cause harm hazardous substances in the implementation of safe work *anything can be a hazard, provided that there is an action or practices in the laboratory. Washing the hands frequently with event occurring soap and water is one of the most important (e.g. swimming the sea; eating in the laboratory) COLOR CODING IN WASTE DISPOSAL COLOR MATERIAL Risk – likelihood of harm taking place; a chance that someone will be harmed by the hazard YELLOW - infectious/pathological specimens (e.g. death due to drowning for not knowing how to swim; (e.g. bandages, cotton, gauze, anything getting diarrhea while eating in the laboratory) with body fluids/parts) LABORATORY SAFETY SYMBOLS Puncture-proof - sharps container in (e.g. broken glassware, syringe, catheter) RED trash bag BLACK - dry, non-infectious material (e.g. paper, shoe covers) GREEN - wet, non-infectious material (e.g. left-over foods) YELLOW WITH - chemical/heavy metals BLACK BAND ORANGE - radioactive wastes 4 AGENCIES FOR LABORATORY SAFETY: 1. Center for Disease Control and Prevention (CDC) 2. Occupational Safety and Health Administration (OSHA) SYMBOL RATING & HAZARD INCLUSIONS 3. College of American Pathologists (CAP) FLAMMABLE 0 – will not burn (water) 4. Joint Commission (JC) SIGN 1 – must be heated for ignition, flashpoint above BIOSAFETY VS BIOSECURITY 93˚C (lube oil, cooking oil) Biosafety – protecting human health and the environment from 2 – must be moderately heated for ignition, the pathologic agent flashpoint below 38 ˚C (diesel fuel) 3 – ignition occurs at most ambient conditions, Biosecurity – protecting the pathologic agent from humans flashpoint below 38 ˚C (gas, acetone) *Note: Biosafety in Microbiological and Biomedical Laboratories 4 – extremely flammable and will readily dispense (BMBL) has become the code of practice for biosafety—the through the air (propane, butane) discipline addressing the safe handling and containment of POISON SIGN 1. venom infectious microorganisms and hazardous biological materials. 2. lead (paints, pencil, batteries) CDC CLASSIFICATION OF INFECTIOUS AGENTS 3. pesticides LEVEL DEFINITION SAFETY EQUIPMENT 4. alcohol (methanol) BIOSAFETY - low risk - laboratory gown RADIOACTIVE 1. gamma radiation - agents with no known potential - gloves 2. sunlight (ultraviolet rays) LEVEL 1 SIGN for infecting healthy people - goggles (BSL-1) 3. x-rays - not known to consistently cause disease in adults BIOHAZARD 1. animal waste - minimal potential hazard to SIGN 2. contaminate PPEs laboratorians 3. laboratory agents - can be performed in an open HOW TO USE THE FIRE EXTINGUISHER? table P – pull the pin in the handle (e.g. Escherichia coli 1655) - agents that are mostly - laboratory coat A – aim the nozzle at the base of the fire BIOSAFETY encountered clinical specimens - gloves S – squeeze the lever slowly LEVEL 2 - microbes pose moderate - goggles S – sweep from side to side (BSL-2) hazards to laboratorians - biological safety HAND WASHING: 40-60 SECONDS - microbes are typically cabinet indigenous (Tuberculosis) and - cuffed lab gowns associated with varying severity - requires the use of biosafety cabinet (e.g. Bacillus anthracis, Staphylococcus aureus, and Streptococcus spp.) BIOSAFETY - agents with potential aerosol - laboratory coat transmission - gloves LEVEL 3 - can cause serious/lethal - goggles (BSL-3) consequence - biological safety - requires the use of fit tested cabinet respirator (N95, N100) - cuffed lab gowns - (e.g. Mycobacterium tuberculosis) - respirator Olivar, Resurreccion, Suaga 1 Clinical Bacteriology Laboratory Notes I BSMLS-3B2 NOSOCOMIAL INFECTIONS 6. Disposal of rejected specimens must be done under the ff - hospital acquired infections conditions: - transmission occurs via healthcare workers, patients, - discussed with diagnostician the reason for rejection hospital equipment or interventional procedures - all concerned individuals are notified of the rejection - bacteria: common cause - stored for 48hours 5 FREQUENT ACQUIRED INFECTION SPECIMEN COLLECTION FROM CLINICAL LABORATORIES 1. Blood Culture 1. Shigellosis - must prepare skin properly with alcohol (70%) and - found in the stool (feces) of infected people, in food or iodine water contaminated by an infected person, and on - tryptic soy broth is used; collect prior fever spike, before surfaces that have been touched by infected people. antibiotic administration - Shigellosis often occurs in toddlers who are not fully - ARD (antibiotic removing device) may be added to blood toilet-trained. culture media to inactivate beta lactam antibiotics (e.g. - fecal-oral route penicillin) an other antibiotics present in the patient - can cause dysentery: a watery stool with blood/mucus blood 2. Salmonellosis - requires at least 2 blood specimens (but no more than - also related to food and water poisoning 3) in a 24-hour period in different sites to rule out - can cause enteric fever (40˚C, chills, body pain) contamination 3. Tuberculosis *Sodium Polyethanol Sulfonate (0.025-0.05% SPS) - caused by a type of bacterium called Mycobacterium - best anticoagulant for blood culture (1st in the order of tuberculosis, it is spread when a person with active TB blood draw). disease in their lungs coughs or sneezes and someone - SPS inhibited: else inhales the expelled droplets, which contain TB  Neisseria sp. bacteria.  Gardnerella vaginalis - aerosol  Streptobacillus moniliformis 4. Brucellosis  Peptostreptococcus - a zoonotic disease (pigs, cows, goats, dogs, rat) - Properties: - transmission: through ingestion of infected milk  Anticomplementary – antibodies produce a products and through inhalation. complement to bind to certain bacteria to destroy - replicate inside neutrophils (immune system will be  Antiphagocytic – prevent WBCs to phagocyte the weakened; WBC becomes predominant) bacteria 5. Hepatitis  Inactivates aminoglycosides - inflammation of the liver  Neutralize bactericidal effect of human serum - can be transmitted through accidental needle prick (Ig is usually neutralized) - Hepatitis B & C: blood-borne transmission *Note: - Hepatitis A: food-borne transmission - ratio of blood culture media in adults: 5:10 COMMON CONSIDERATIONS IN SPECIMEN COLLECTION - ratio of blood culture media in neonates: 1:10 1. Adequacy - blood culture is kept for 7days (routine) before reporting - amount collected should be sufficient as no growth - sufficient amount allows repetition of test procedures - Signs of Growth: 2. Proper site  Turbidity – cloudiness in the water - collected at area where infection is suspected using  Hemolysis – destruction of RBCs aseptic (sterile/clean) technique  Clot/Pellicle formation – presence of clots 3. Proper time  Bubbles – due to bacteria that produces gas - collect specimen at the right stage of disease  Colonies in top of blood layer 4. Promptness of examination 2. Throat and Nasopharyngeal Cultures - should be taken immediately to the laboratory and - collected on a cotton-tipped applicator and applied to a process instantaneously slide or culture medium 5. Specimen collected before administration of antimicrobials - normal flora would include Alpha hemolytic - because taking an antimicrobial may cause false no streptococcus (S agalactiae) and Streptococcus growth result pyogenes - if the specimen has been taken after initiation of - most common pathogen: Group A Beta Hemolytic Strep antimicrobials, use of antibiotic removing device or Streptococcus pyogenes specimen dilutions is suggested 3. Sputum Culture CRITERIA FOR SPECIMEN REJECTION - collected from deep cough, at least 5ml for culture 1. Insufficient information - for MTB culture, addition of N-Acetyl-L-Cysteine - mislabeling (digestion and for the sputum to have a recommended - regarding the specimen, unidentified, improperly consistency), and 2% sodium hydroxide (NaOH) labeled specimen; improper (decontamination) - container (particularly anaerobic cultures) - acceptable specimens upon microscopic view should 2. Insufficient amount have less than 10% epithelial cells/LPF and greater than - exact amount is needed 25 PMNS/LPF 3. Grossly contaminated specimen and dry swabs for culture *PMNs – polymorphonuclear cells 4. Delay in collection and transport in the laboratory *Cary Blair: 5. Only process 1 specimen if there are 2 specimens sent even - transport medium swab for stool and rectal swab if they are in different containers - a simple, semi-solid, non-nutritive medium used for the *Note: specimen that are not processed due to criteria for collection and preservation of microbiological rejection should be stored in a separate locker (e.g. refrigerator) specimens. for several days (maximum of 48hours) until such time notice - it is used for the transportation of clinical specimens was discussed with the requesting diagnostician, then disposal suspected to contain enteric pathogens, including must be done to contained contamination according to the Shigella, Salmonella, Vibrio cholerae, and Escherichia Institutional Biosafety guidelines. coli O157: H7 Olivar, Resurreccion, Suaga 2 Clinical Bacteriology Laboratory Notes I BSMLS-3B2 4. Urine Culture MODULE 2 - collected via midstream clean catch urine or suprapubic MICROSCOPY aspiration Latin word “micro”: small - culture within 2 hours after collection Greek word “skopos”: to look at - may be stored or refrigerated (4°C as long as 24hours Microorganisms are usually measured in: if not processed immediately)  micrometers (um) – 10-6 m *Suprapubic Aspiration:  nanometers (nm) – 10-8 m - maybe stored or refrigerated at 4°C for 24hrs TYPES OF MICROSCOPE - 20° aspiration of urine from bladder o According to the number of Lenses - colony count: 1 x 105 CFU/ mL indicates UTI 1. Simple Microscope 5. Stool Culture – has a single biconcave lens - specimen should not be contaminated with urine – usually has a 10x magnification - collect during early phase of the disease, before 2. Compound Microscope administration of antimicrobial – has two or more lenses and is classified based in - amount needed: 1-2g quantity of stool is sufficient illumination - storage: at 2-8°C if not processed immediately *Note: 6. Genital Culture - the highest magnification you can get is 1000x - neisseriae are inhibited by fatty acids and salts, o According to Illumination therefore calcium alginate and cotton swabs should not 1. Light Microscope be used for specimen collection – employs visible light as source of illumination - no usage of cotton swabs because it contains fatty acids 2. Electron Microscope and salts – employs beam of electrons as source of illumination - recommended use of Dacryon, Rayon and polyester- – used to magnify viruses tipped swabs TYPES OF LIGHT MICROSCOPE 7. Cerebrospinal Fluid 1. Brightfield Microscope - almost 3-10mL of CSF is collected and is done by – objects appear dark against a bright background physician – visible light is passed through specimen and then through - be processed immediately, if delayed store in incubator a series of lenses that bend the light in a manner that results - do not refrigerate CSF in magnification of the organism - collected in 3 tubes: – commonly used for visualization of bacteria, parasites,  Tube 1 for Chemistry and Serology and fungi  Tube 2 for Microbiology – Magnification:  Tube 3 for Hematology  Ocular lenses: 10x *Note: – Types of Objective Lens If there’s a 4th tube, it will be submitted to the Microbiology  Scanner: 4x section)  LPO: 10x (yellow) The CSF for microbiology is collected 2nd to avoid  HPO: 40x (blue) contamination of the microflora in the skin or brain  OIO: 100x (gray) *Note: NORMAL FLORA - to solve for total magnification: Skin Staphylococcus, Micrococcus, = ocular lens (10x) x objective lens magnification Streptococcus, Propionibacterium, - when viewing in LPO, use the course adjustment knob Corynebacterium, Candida, Malassezia - in HPO, make sure that the specimen is already visible in LPO, then use the fine adjustment knob to further Oral cavity Streptococcus, Neisseria, Veillonnella, magnify Lactobacillus, Bacteroides, Treponema, Hemophilus, Candida, Diphteroids Large Intestine Bacteroides, Clostridium, Streptococcus, Staphylococcus, Enterococci, Escherichia, Proteus, Enterobacter Genital Tract Lactobacillus, Staphylococcus, Candida, Streptococcus Urinary Tract Staphylococcus, Streptococcus, Lactobacillus, Diphtheroids, Candida Normally sterile sites:  All internal organs (except appendix and large intestine)  Body fluids  Blood 2. Darkfield Microscope  Urine in bladder, ureters and kidneys – objects appear bright against a dark background – uses a darkfield condenser – routinely used to observe:  Treponema pallidum “Spirochetes” – causes syphilis, a sexually transmitted disease causing genital ulcer or lesion (chancre) Chancre – painless lesion that has even edges  Cryptococcus neoformans – an opportunistic fungus that causes disease in HIV positive patients Olivar, Resurreccion, Suaga 3 Clinical Bacteriology Laboratory Notes I BSMLS-3B2 HANDLE AND CARE OF THE MICROSCOPE 1. Carry the microscope in upright position with two hands, one hand supporting the base & the other hand grasping the arm. 2. Place the microscope carefully on the working table, about 1 inch away from the table edge. 3. Remove the dust from the microscope using soft brush (preferably camel’s hair) or you may blow it away. Only after this should the lenses be cleaned with lens paper. 4. Clean the eyepiece lens with dry lens paper and the objective 3. Phase Contrast Microscope lenses with lens paper or cotton (dry or moistened with xylene – observes unstained or 95% ethyl alcohol). specimen where light 5. If the microscope is equipped with electric cord, check if it is refracted by cells are in good condition before plugging into the outlet. different from light 6. Make sure that the scanner or the low power objective (LPO) refracted by surrounding is in focusing position. Always start each new observation at medium. Thus, contrast scanner or LPO. is increased making MANIPULATION OF THE MICROSCOPE more visible 1. Place the prepared slide carefully on the stage and secure it – enhances appearance of colorless or transparent with stage clips or brackets. organism 2. Observe under OIO, draw and describe the morphological – used in wet mounts, allows observation of viable appearance of the bacteria using the different prepared slides. specimen METHODS OF IDENTIFYING BACTERIA – primarily used in visualization of important fungi 1. Living State Bacteria – to differentiate true motility from Brownian movement (false motility of a bacteria that 4. Fluorescence Microscope appears stable) – with built in UV light which strikes dyes and pigment  Wet mount preparation substances emitting longer wavelength  Hanging drop preparation – causes organism to glow against a dark background – uses biconcave slide – used in immunodiagnostics, demonstrate antibody – provides larger area due to depress biconcave space stained with fluorescent dye combining with specific – petroleum jelly, a sealant making the specimen antigens secure – light source at the side (+) result: directional purposeful motility – dyes used: fluorophores or fluorochromes which absorbs (-) results: random jiggling or shaking remaining in the UV light same position *Quenching/Photobleaching 2. Fixed State Bacteria – for staining – fading of fluorescence due to exposure to light energy BACTERIAL STRUCTURES – can be prevented by storing fluorescent slides in a dark container and refrigerate at 2-8˚C 1. Capsule – not common to all bacteria – a slimy gelatinous are around the cell wall – antiphagocytic, higher resistance to phagocytosis (e.g. Strepcoccus pneumoniae, Neisseria meningtides, TYPES OF ELECTRON MICROSCOPE Haemophilis influenza) 1. Scanned Probed Electron Microscope *Neufeld Quellung Test – capsular swelling test used to – produces 3D image of the cell and structural surfaces identify bacteria with capsules – major capital investment and is not needed for the 2. Cell Wall laboratory diagnosis of most infectious diseases (except for – common to most bacteria certain viruses and microsporidian parasites) – determines gram positivity (blue to purple) and negativity 2. Transmission Electron Microscope (pink to red) – produces 2D image of the cell structure – defines the shape/contents of the bacteria *E. coli – gram – usual site of antibiotic action negative – peptidoglycan: main component  Thick peptidoglycan = gram positive  Thin peptidoglycan = gram negative *Mycoplasma and Ureaplasma – organisms without cell wall; they are commensals *Note: - the cell wall is the target by the antibiotic - commensal (living with benefits without hurting the host) in human being - normal flora (living without causing disease) but can cause disease when expose to antibiotics Olivar, Resurreccion, Suaga 4 Clinical Bacteriology Laboratory Notes I BSMLS-3B2 3. Plasma Membrane CLASSIFICATION OF BACTERIA – surrounds the cytoplasm ACCORDING TO THEIR FLAGELLA – site of energy production (ATP) – allows the transport of nutrients in and out of the cell 4. Pili or Fimbriae – common among gram positive bacteria (e.g. E. coli, N. gonorrhea) *Sex pili – transfer of genetic material or gene conjugation, for reproduction *Common pili – attachment to host cell; infectious 5. Endospores – resistant structures, allow bacteria to survive in adverse conditions o Monotrichous – single flagellum at one end – not found in all bacteria o Amphitrichous – single flagellum each at both ends – contains calcium dipicolinate or dipicolinic which is o Atrichous – no flagella responsible for resistance o Lopotrichous – tuft of flagella at one or both ends (e.g. bacillus, clostridium) o Peritrichous – surrounded by flagella 6. Flagella o Cephalotrichous – several flagella at both ends – locomotory organ – common among bacilli and spirilla BACTERIAL MORPHOLOGY – rare among cocci o The largest bacterium known, Thiomargarita namibiensis, *Note: is found in ocean sediment and generally has a diameter of - Motile bacteria: E. coli, Salmonella typhosa 0.1 to 0.3 mm. - Non motile bacteria: Klebsiella pneumonia, Shigella o Most bacteria range in size from 0.4 to 2 µm dysenteriae o Individual bacteria may form characteristic groupings - Motility is best seen at 25˚C o Bacteria occur in three basic shapes: *Axial Filaments/Periplasmic Flagella  Cocci – spherical or ovoid – locomotory organ of Spirochetes  Bacilli – rodlike 7. Metachromatic Granules  Spiral – corkscrew or curved – food reserves – much granules = mycobacterium tuberculosis BACTERIAL ARRANGEMENT – Babes Ernst granules = corynebacterium diphtheria o Pairs, chains, clusters or other groupings; such formation 8. Nucleoid is usually characteristic of a particular species – contains DNA found in the center of the bacteria o Coccus arrangements are determined by their orientation 9. Plasmid and degree of attachment at the time of cell division – carries antibiotic resistant gene o Bacillus and Spiral arrangements are less medically 10. Ribosome important – site of protein synthesis – gives granular appearance of bacteria under electron microscope WAYS TO DEMONSTRATE MOTILITY 1. Semisolid Media – SIM (Sulfure Indole Motility) 2. Use of Flagellar Stains  Fisher  Gray and Leifson 3. Hanging Drop Motility Test (Inverted or Biconcave slide) – a special type of wet mount (in which a drop of medium containing the organisms, is placed on a microscope slide) often used in a dark illumination to observe the motility of a bacteria – a drop of cultured bacteria is placed on a coverslip that is surrounded by petroleum jelly, and inverted over the well of a depression slide. The drop of cultured bacteria hangs from the coverslip, and the petroleum jelly forms a seal that prevents evaporation of the sample Olivar, Resurreccion, Suaga 5 Clinical Bacteriology Laboratory Notes I BSMLS-3B2 MODULE 3 STEPS IN A TYPICAL CULTURE TO SMEAR TRANSFER SMEAR PREPARATION 1. Flame the inoculating loop Aseptic Technique 2. Flame the mouth of the tube while holding the cap – refers to a procedure that is performed under sterile 3. Remove some bacteria using the loop conditions 4. Flame the mouth of the tube and replace the cap – includes medical and laboratory techniques which deal with 5. Flame the mouth of the tube to which the bacteria are being cultures and human cells and tissue for transplantation transferred *Note: to avoid contamination, use aseptic techniques (sterile) 6. Inoculate the tube with bacteria from the loop OBJECTIVES 7. Flame the mouth of the tube and replace the cap To prevent the access of micro-organisms during the 8. Flame the inoculating loop preparation and testing SMEAR PREPARATION - contaminants may cause false growth of microorganisms - Most bacteria have no color, so USES OF ASEPTIC TECHNIQUE they generate little contrast in o prevent contamination of the specific microorganism we the microscope field. are working with - Therefore, to see bacteria with o prevent contamination of the room and personnel with the the microscope, it is necessary microorganism we are working with to apply color through staining SOURCES OF CONTAMINATION - In order to observe morphology 1. Atmosphere – collect samples from the air via swabbing is of the bacteria under the applicable when ill patients are enclosed in a certain place microscope, a bacterial smear 2. Breath must first be prepared before 3. Hands proceeding to staining. 4. Clothing SMEARING 5. Hair - Is a simple technique that involves the placement of a small 6. Working surface sample of the microorganism on a slide, dispersing it, then 7. Equipment drying/heating it to fix or immobilize the cells onto the slide PROCESSES INVOLVING ASEPTIC TECHNQUE surface. o Sterilization – a process by which articles, surfaces, or - Preparing the smear requires attention to a number of medium are made free from all microorganisms either in details that help prevent contamination of the culture & the vegetative or spore state ensure safety of the worker. o Disinfection – a process by which articles, surfaces, or - This procedure also kills the cells. medium are made free from all pathogenic microorganisms - Dispersing and drying the cells enhances the uptake of the (organisms capable of giving rise to infection) dye used in staining, while heat fixing is required to prevent o Antiseptic – the process by which growth of bacteria is cells from being washed off the slide during staining and inhibited but not killed subsequent rinsing. What needs to be Sterilized in Aseptic Technique? STEPS IN SMEARING 1. Culture media 1. Placement of a small sample on a slide 2. Fluids used in the laboratory 2. Dispersing it – enhances the uptake of the dye used in 3. Reagents staining 4. Lab containers 3. Drying or heating to fix or immobilize the cells unto the 5. Lab equipment surface GENERAL PRINCIPLES OF ASEPTIC TECHNIQUE Bacterial Smear 1. Disinfect the work area before – a thin layer of bacteria placed starting or performing to reduce on a slide for staining potential contaminants on the – to determine the right amount, bench top, and disinfect again after compare the sample with the to protect others from possible newspaper print contaminations  Too thick – the organism - clean or disinfect the glass of the biosafety cabinet may not be visible first, then the surface, and sides.  Too thin – the organism 2. Flame the inoculating loop before may be too few or absolute and after transferring of bacteria FIXATION from one container to another. Fixation – a process of making the smear adhere to the slide - never lay an inoculating loop Ways of Fixing the Smear Sample: on the bench top if you are not sure it has been flamed 1. Passing over a flame first. When in doubt, flame the loop. – using Bunsen burner or electric flame 3. Flame the opening of glass 2. Placing the slide in an oven containers before and after – 65-75 degree Celsius removing the bacteria. Likewise, 3. Immersing the smear in a chemical fixative flame the opening the tube before – ethanol or methanol and after transferring the bacteria to a container. - opening of the tube is susceptible to exposure of possible contaminants 4. Do not lay the cap of the containers on the bench top while the bacteria are being removed and transferred to the container. The cap should remain under your control throughout the transfer. 5. Work quickly and efficiently to minimize the time of the culture being exposed to the environment. Olivar, Resurreccion, Suaga 6 Clinical Bacteriology Laboratory Notes I BSMLS-3B2 LIQUID CULTURE MEDIA 8. Apply the smear to the slide evenly in a circular motion, BACTERIAL SMEAR PREPARATION from inner to outer region. 1. Prepare PPE, materials, equipment, and waste disposal needed for the activity. – Bunsen burner, inoculating loops, slides, etc. 9. Flame the mouth of the tube before capping. 2. Disinfect workspace and place materials in the BSC. 10. Flame the inoculating loop until the wire turn bright red. Allow it to cool and set it aside. 3. Prepare slides by passing it over the flame and making a margin on one side. Label properly. 11. Air dry the smear samples. Heat the fixative in preparation for staining. 4. Gently shake the tube to resuspend the microorganisms. Avoid moistening the cap. 12. Disinfect the workspace and dispose wastes properly. 5. Flame the inoculating loop until it reached the bright red color, and allow it to cool. 6. Hold the inoculating loop. Remove cap and do not place it on the working area. Flame the mouth of the tube to avoid contamination. 7. Fish out a loopful of specimen. Olivar, Resurreccion, Suaga 7 Clinical Bacteriology Laboratory Notes I BSMLS-3B2 SOLID CULTURE MEDIA 8. Apply the smear to the slide evenly in a circular motion, BACTERIAL SMEAR PREPARATION from inner to outer region. 1. Prepare PPE, materials, equipment, and waste disposal needed for the activity. – NSS, Bunsen burner, inoculating loops and needles, slides, etc. 9. Flame the inoculating loop until the wire turn bright red. Allow it to cool and set it aside. 2. Disinfect workspace and place materials in the BSC. 10. Air dry the smear samples. Heat the fixative in preparation for staining. 3. Prepare slides by passing it over the flame and making a margin on one side. Label properly. 11. Disinfect the workspace and dispose wastes properly. 4. Place a drop of sterile NSS at the center of the margin area of the slide. 5. Flame the inoculating loop until it reached the bright red color, and allow it to cool. 6. Using the inoculating needle, fish a single, isolated colony from the sample. 7. Transfer the colonies by touching onto the sterile NSS on the slide. Olivar, Resurreccion, Suaga 8 Clinical Bacteriology Laboratory Notes I BSMLS-3B2 MODULE 4 GENERAL RULES IN GRAM STAINING STAINING TECHNIQUES 1. ALL COCCI ARE GRAM (+) EXCEPT Purpose of Staining: Neisseria, Bramhamella, Veilonella, Acidaminococcus and  used to color microbes with dyes that emphasize certain Megasphera structures 2. ALL BACILLI ARE GRAM NEGATIVE EXCEPT  used to appreciate the appearance and morphology of Mycobacterium, Clostridium, Corynebacterium, Bacillus, bacteria Erysipelothrix, Lactobacillus and Listeria – for easier identification 3. ALL SPIRALS ARE GRAM NEGATIVE IN PRINCIPLE  used to differentiate one group of organism from another  – to properly diagnose a disease *Note: CLASSES OF IONIZABLE DYES Most of the cocci are gram positive while bacilli are mostly gram 1. Basic dyes negative - common dyes used in bacteriology Lipid content in the cell wall of a gram positive stain is not easily - cationic – positively charged dyes that binds to negative cell penetrated by the decolorizer components (e.g. cell wall) Gram positive – CV dye is added to the sample penetrating the *Examples: cell wall, then the mordant dye is added, next is the decolorizer  methylene blue dye which will retain the crystal violet dye due to its less  basic fuchsin penetration to the cell wall, and safranin is finally added which  crystal violet will be absorbed by the sample and is notable to enter the cell  malachite green wall giving purple or blue color  safranin Gram negative – CV dye is added to the sample penetrating the 2. Acidic dyes cell wall, then the mordant dye is added, next is the decolorizer - anionic – negatively charged dyes that binds to positive cell which will consume or decolorize the crystal violet dye due to components its greater penetration to the cell wall, and safranin is finally *Examples: added penetrating the sample appearing in pink color  eosin  rose bengal GRAM STAINING REACTION THEORIES  acid fuchsin STAINING TECHNIQUES 1. Simple Staining - uses a single basic dye - visualize entire microbes, cell shapes and structure *Examples:  methylene blue  crystal violet  carbolfuchsin  safranin 2. Differential Staining - uses more than one dye – to divide the bacteria for differentiation COMPONENTS OF GRAM STAINING - divides bacteria into separate group 1. Primary stain *Examples: - crystal violet, methyl violet, gentian violet  gram staining – positive and negative 2. Mordant  acid fast staining – differentiate acid fast bacilli to non-acid – enhances the effect of dye used fast bacilli - Gram’s iodine 3. Diagnostic Antibody or DNA probe – mediated 3. Decolorizer - used for the identification of a specific organisms – retain more primary stain due to permeability *Examples: - ethanol, acetone, or 1:1 ethanol acetone mixture  Chlamydia trachomatis 4. Counterstain  B. pertussis - dilute carbolfuchsin, safranin, or neutral red *Note: DNA is being destroyed to have a single stranded DNA, then a PRINCIPLE OF GRAM STAINING probe is used as markers that will attach to the single DNA o bacteria with thick cell walls (composed of thicker strand, after the attachment, it will be stained and allows DNA peptidoglycan) containing teichoic acid retain the CV-iodine identification. complex dye after decolorization and appear blue, thus they 4. Negative Staining are gram positive - used to demonstrate the presence of diffuse capsule o bacteria with thinner cell walls containing LPS (with less to surrounding some bacteria none teichoic acid) do not retain the dye complex and - the capsule of the fungi would appear white appear pink, thus they are gram negative (e.g. C. neoformans) *Examples:  1. India ink  2. nigrosin dye GRAM STAINING - developed by Hans Christian Gram - classifies bacteria into two large groups - primarily used to discriminate between two bacteria that causes disease in the lungs Olivar, Resurreccion, Suaga 9 Clinical Bacteriology Laboratory Notes I BSMLS-3B2 PROBLEMS ENCOUNTERED IN GRAM STAINING AND HOW TO SOLVE THEM PROBLEM CAUSES Nothing visible on the - smear too thin slide - slide was wiped during drying process – repeat the process - smear was not heat fixed – fixation must adhere to the slide, thus if it is not fixed, it will cause washing out of the semear Stain precipitate - unfiltered stain was used *Note: present – due to – filter paper is used for Acid fast stain Kinyoun’s method is a cold method since heat is prolonged storage, and siltration not used but a higher concentration of carbolfuchsin (20%) is erroneous process used as a primary stain. Gram (+) cells appear - removal of MgRNA gram negative - non-viable organism MODIFICATION OF ACID-FAST STAINING (gram variable) 1. PAPPENHEIM’S METHOD - technical/human error in - used to differentiate M. smegmatis from M. tuberculosis staining procedure - M. smegmatis – found in smegma at the penile area of males Gram negative - presence of safranin crystals appearing yellowish and in popcorn shape organism looks like 2. BAUMGARTEN’S METHOD needs than rods - used to diiferentiate M. leprae from M. tuberculosis Gram negative cells - underdecolorization - Leprosy – decaying body with loss of feeling appear gram (+) - prolonged staining – CV is no longer removed TB – DOTS by the decolorizer - tuberculosis – directly observed treatment, short course - technical error - a 6-8 months long program which aims to diagnose, treat and Mixture of Gram (+) - smear is too thick prevent the spread of TB and Gram negative - culture is contaminated from a pure culture TB DOTS TEST 1. TUBERCULIN TEST ACID FAST STAIN - basic screening tool - a tool for identification of Mycobacterium tuberculosis - skin test - used to stain bacteria that have high lipid content in their cell - inflammation in the skin with a diameter of 10 mm or more wall - used for patients less than 15 years old PRINCIPLES OF ACID-FAST STAIN 2. TB CULTURE - the primary stain binds to mycolic acid in the cell walls of the - using solid and liquid media mycobacteria and is retained after decolorization with acid - Solid media alcohol – Lowenstein-Jensen media, a protein rich media coming - the AFB stain pink, straight or slightly curved rods, sometimes from eggs a beaded appearance in a blue background – Ogawa media - Liquid – Middlebrook series 3. RAPID MOLECULAR TEST (XPERT MTB/RIF ASSAY) - rapid test that detects M. tuberculosis and Rifampicin resistance - Rifampicin – a core antibiotic used to treat TB RESULTS INTERPRETATION T MTB detected, Rifampicin resistance not detected RR MTB detected, Rifampicin resistance detected TI MTB detected, Rifampicin resistance indeterminate N MTB not detected I Invalid *Note: 4. DIRECT SPUTUM SMEAR MICROSCOPY (DSSM) Acid fast stain Ziehl-Neelsen method is a hot method with a - primary diagnostic method primary staining of 10 minutes, application of heat (mordant), - provides a definitive diagnosis of active TB and until the smoke arises, acid alcohol (decolorizer) is applied. COLLECTION OF SPUTUM Acid-alcohol is a combination of HCl and ethanol application of  two specimens are required methylene blue (counter stain)  spot – same day same time collection  morning collection – next day early morning  spot-spot – collection after an hour  at least 1 teaspoon full (5-10ml) of specimen is needed Olivar, Resurreccion, Suaga 10 Clinical Bacteriology Laboratory Notes I BSMLS-3B2 PROCEDURE FOR SPUTUM COLLECTION MODULE 5 1. Instruct patient to rinse mouth with water STERILIZATION 2. Breathe deeply, hold breath for 1-2 seconds, and exhale - a sterile surface or object is completely free of living slowly. Repeat sequence twice. microorganism and viruses. 3. Cough strongly after inhaling deeply for the third time and - kills all either pathological or non-pathological try to bring up sputum from deep within the lungs microorganisms 4. Expectorate sputum in the sputum cup - methods used include heat, ethylene oxide gas, hydrogen 5. Examine the specimen to see that it is not just saliva peroxide gas, plasma ozone and radiation - the probability of a microorganism, surviving on an item DSSM RESULT INTERPRETATION subjected to treatment is less than one in one million (Sterility Assurance Level) DISINFECTION - eliminates most pathogens but not necessarily all types of microbes - reduces the level of contamination - chemical disinfection does not kill spores, unlike chemical sterilization. - some common laboratory disinfectants include freshly prepared 10% bleach and 70% ethanol DECONTAMINATION - renders an item or material safe to handle. - the level of microbial contamination is reduced enough that it can be reasonably assumed free of risk of infection transmission - sterilization, disinfection, and antisepsis are forms of decontamination ANTISEPSIS - the application of a liquid antimicrobial chemical to skin or living tissues CLASSIFICATION OF TB - includes swabbing an injection site on a person or animal, 1. BASED ON BACTERIOLOGICAL STATUS and handwashing with germicidal solution - Bacteriology confirmed, or clinically diagnosed PHYSICAL METHODS OF STERILIZATION - X-ray or DSSM positive 1. Scrubbing with soap and water 2. BASED ON ANATOMICAL SITE 2. Filtration - pulmonary (lungs) TB or extra pulmonary (eyes, bones) TB - used to sterilize antibiotic solution and vaccines 3. Sedimentation FIRST-LINE DRUGS AGAINST TB - process of settling or being deposited as a sediment  Pyrazinamide 4. Low temperature  Rifampicin - 5 degrees Celsius for refrigerator temperature  Isoniazid - 0 degrees Celsius or subzero freeze drying through  Ethambutol sublimation  Streptomycin 5. High temperature  Pasteurization 3. BASED ON DRUG SUSCEPTIBILITY - process of heating and cooling food to kill bacteria DRUG SUSCEPTIBILITY DESCRIPTION - LTH (low temperature holding) = 62.7˚C for 30 minutes Resistance to 1 first line anti-TB - HTSH (high temperature short time) MONORESISTANT – TB drug = 71.1˚C for 15 seconds  moist heat Resistance to more than 1 first - destroys microorganism by the irreversible POLYDRUG – line anti-TB drug denaturation of enzymes and structural proteins, RESISTANT TB with the amount of water present - boiling = 100˚C for 5 minutes Resistance to atleast both anti- - free flowing steam MULTIDRUG – TB drug - autoclave = 121˚C at 15lbs psi for 15 minutes or RESISTANT TB 132˚C - Tyndallization/Fractional Sterilization = 100˚C for 30 Resistance to any minutes for 3 days or 60˚C for 5-6 days EXTENSIVELY DRUG – fluoroquinolone and to at least - Inspissation: used to sterilize culture media rich in RESISTANT TB one of three second line proteins; 70-80˚C for 2 hours for 3 days injectable drugs  dry heat Resistance to rifampicin detected - used to sterilize items that might be damaged by RIFAMPICIN – using phenotypic or genotypic moist heat or that are impenetrable to moist heat RESISTANCE TB methods (e.g. powders, petroleum products, sharp instruments) - accomplished by conduction - heat passing through a flame - hot air/oven baking = 160˚C for 2 hours; 171˚C for 1 hour; 121˚C for 16 hours - Incineration = 300-400˚C - Cremation = 400-1000˚C for burning dead bodies Olivar, Resurreccion, Suaga 11 Clinical Bacteriology Laboratory Notes I BSMLS-3B2  radiation - kills germs that can cause disease and neutralizes other harmful organisms - non-ionizing (UV) = longer wavelength, low energy - ionizing = shorter wavelength, high energy AUTOCLAVE - pressure cooking - common method for moist sterilization - effective in killing fungi, bacteria, and viruses but does not necessarily eliminate prions or viruses CHEMICAL METHOD OF STERILIZATION GASEOUS STERILIZATION  ethylene oxide  Formaldehyde  Nitrogen dioxide  ozone LIQUID STERILIZATION  alcohol (ethanol isopropanol)  halogens (chlorine, sodium)  phenolic compounds  detergent (anionic, QUATS)  glutaraldehyde  acids  heavy metals Olivar, Resurreccion, Suaga 12