Unit 4 Microbiology_1.0.pptx

Microbiology The study of microorganisms 2.3,3.1.1,3.1.6,3.2.2,3.3.1,3.3.3,3.3., 3.4.7,4,4,1, 6.1.4 1 Objectives Microbiology  Objectives 1. Basis definitions 1.1 Define basis def...

Microbiology The study of microorganisms 2.3,3.1.1,3.1.6,3.2.2,3.3.1,3.3.3,3.3., 3.4.7,4,4,1, 6.1.4 1 Objectives Microbiology  Objectives 1. Basis definitions 1.1 Define basis definitions used in microbiology 2. Different types of microorganisms 2.1 List the four different types of microorganisms 3. Collecting methods used in microbiology 3.1 Lists some collecting methods used in microbiology 4. Amies Transport Media and its modifications 4.1 Describe Amies Transport Media and its modifications 5. Normal flora 5.1 List the specimens that have normal flora 6. Specimens that are sterile 6.1 List the specimens that are sterile 3  Objectives 7. Basic procedure in microbiology 7.1 Describe the basic procedure in microbiology 8. Different types of media 8.1 Describe the different types of media 9. How to make media 9.1 Describe the step-by-step procedure in making media 9.2 List the quality control used in making media 10. Different categories of media 10.1 List and describe the different categories of media 10.2 Describe the order of inoculating me 4  Objectives 11. Setting up cultures 11.1 Describe the procedure on how to set up cultures 12. Isolated colonies 12.1 Describe the reason for isolated colonies 12.2 Describe the step-by-step procedure in streaking plates 13. Bacterial Physiology 13.1 Describe some basics bacterial physiology 14. Different environmental jars 14.1 Describe the step-by-step procedure in setting up an anerobic jar 14.2 Describe the step-by-step procedure in setting up microaerophilic jar 14.3 Describe the step-by-step procedure in setting up a candle jar 5  Objectives 15. Growth Curve 15.1 Describe in detail the growth curve 16. Different body site 16.1 Describe in detail what media to inoculate for different body sites 16.2 Describe in detail how to collect blood cultures 17. Stains 17.1 List the most common stain used in microbiology 17.2 List other stain used in microbiology 6  Objectives 18. Gram stain 18.1 Describe the step-by-step procedure in using the Gram stain 18.2 Describe the different reactions in the Gram stain 19. Antimicrobials 19.1 Define antimicrobials 19.2 Define broad spectrum 19.3 Define narrow spectrum 19.4 Describe the step-by-step procedure in preparing the disc and strip diffusion method 7  Objectives 20. Storage of microbiology specimens 20.1 Describe on how to store microbiology specimens 21. Specimen rejection 21.1 Describe the criteria for specimen rejection 8  Discovery The chance observation in 1881 of bacteria growing on the surface of a spoiling slice of boiled potato led Robert Koch to devise the technique called single colony isolation He then developed media and agar so single colonies can be isolated to determine what kind of bacteria is present 9 Definitions Acid – fast bacilli ( AFB) – Bacteria that maintains its color after treatment with acid solutions. E.g. tuberculosis, leprosy Acid – fast stain – a special acid base stain used to identify AFB. E.g. Ziehl- Neelson stain Aerosol – a spray of fine particles in the air 10 Agar – an extract of seaweed used to grow bacteria Agar plate – a sterile petri dish in which agar is solidified. Agar slant – a tube in which agar is allowed to solidify at an angle. Anaerobe – an organism that grows without O2 11  Aseptic technique – a routine used to prevent contamination and to maintain sterility Bacillus – rod shaped bacteria Bacteria – one celled microorganisms Bacteriology – study of bacteria. Chlamydia – tiny bacteria responsible for causing a sexually transmitted disease 12 Bacillus 13  Coccus – a spherical-shaped bacteria Colony – a mass of bacteria growing on a media Culture – the act of growing bacteria on a media Culture and Sensitivity ( C&S) – the act of growing a microorganism and testing to determine which antibiotic is effective 14 Coccus 15  Gram neg – bacteria that stains pink/red with a gram’s stain Gram pos – bacteria that stains blue/purple with a grams stain Gram’s stain – crystal violet, iodine, alcohol-acetone & safranin. Used to differentiate between gram + and gram - bacteria 16  Inoculation – the introduction of microorganisms to media Inoculation loop – a wire with a loop at one end and a handle on the other, heated and used to inoculate or transfer bacteria. Calibrated loop – a loop calibrated to 1 microliter used to plant urines. Loop must be placed in a vertical position in the urine and then inoculated onto media 17  Medical Microbiology – the study of living microorganisms that can cause infection and/or disease in humans Media – a solid or liquid substance containing nutrients on which to grow bacteria Mordant – a substance that can cause dye to stick to an object, e.g. iodine 18  Mycology – the study of fungi Normal Flora – bacteria normally found in the body, e.g. staph on the skin, E. coli in the colon. Parasite – an organism that lives off another living creature, e.g. tape worm Parasitology - the study of parasites 19 Pathogens – disease causing microorganisms Petri Dish – flat plastic plate with a cover used to hold media Septicemia - is the presence of microorganisms in the blood 20 Spirilla– spiral shaped microorganisms Sputum – mucous secretions from the lungs Transport media – used to transport microorganisms to the lab to be cultured. Keeps the microorganisms alive so they can be cultured. 21  Virology – the study of viruses Microbiology Department – the section of the lab that identifies organisms which cause illness. Nosocomial infection – infection spread in a health care facility, e.g. E.coli, staph, Candida albicans. Also called Healthcare- associated infection (HAI) 22  Microbiology studies: Bacteria Fungi – Candida albicans, molds Parasites – worms, Malaria, amoebas, Trichomonas vaginitis Rickettsiae – tiny bacteria that resemble both bacteria and viruses. ( have the ability to change shape) They cannot survive outside the cell of a host, e.g. Typhus, Rocky Mountain Spotted Fever. 23  Viruses – smaller than bacteria. Very difficult to grow and treat. 24 25 26 Spores – spores are robust and metabolically dormant structures that are produced by a process of sporulation to prevail over harsh and unfavorable climatic conditions of starvation and stress. They are formed by microorganisms Bacterialspores can survive drought, extreme temperatures, and low pH. Once favorable conditions return, the protective proteins dissolve 27 Anthrax spores 28  Collecting Microbiology specimens Stool for C&S – must be collected in a sterile container Stool for O&P – must be collected in SAF Sputum for C&S – sterile container Urine for C&S – sterile container Throat swabs Blood cultures Wound swabs 29 Patient instructions for C&S  Please check the expiration date on the  container.  If expired, get new container from the lab  First and last name  Provincial Health Care Number and Date of Birth  Date and time of collection  Write time and date of collection on requisition  Do not use laxative, enemas, or antibiotics for one week prior to collection of specimen 30 Amies Transport Media  Amies medium with inorganic buffer ensures maintenance of microorganisms without overgrowth. This transportation system consists of a sterile swab and transport media 31 Procedure 1) Collect specimen with the sterile swab 2) Insert sterile swab into the media 32 Amies Transport Media 33 Amies Transport Media with charcoal Charcoal helps to neutralize materials that are toxic to sensitive pathogens like Neisseria gonorrhoeae Therefore , when collecting specimens for GC use Amies Transport media with charcoal 34 Amies Transport Media with charcoal 35 Cary Blair Transport Medium This is a semi solid medium use in the transportation of stool and rectal swabs 36 Specimens that have normal flora Throat Nose Ear Vaginal Sputum Stool specimens The technologist must differentiate the normal flora from the pathogens 37 Sterile Specimens Blood CSF Fluids from pleural, peritoneal, and joint cavities Surgical tissues Any bacteria found in these specimens, are identified as pathogens. 38 Procedures in a microbiology lab 1. Collect the specimen 2. Stain – to highlight the organism 3. Examine slide under the microscope 4. Inoculate – on various media 5. Incubate - 37°C incubator 6. Identify and further isolate, do biochemical tests to help with identification 7. Antibiotics – using sensitivity discs 39 Making a smear To make a smear from a swab, roll the swab across the slide, leaving a thin layer of material. Air dry Heat fix by using a heating block heat block ( 37°C) or use ethanol 40 MEDIA 41 Media Consists of amino acids, sugars, minerals, salts and vitamins May also contain cooked blood, meat, milk, etc. ( food to enhance bacterial growth) It may be solid, liquid or semi solid Solid – to examine the shape, size and color Liquid – examined for the production of gas, odor, or change in PH 42 Agar Isa seaweed extract. Liquid when heated and solid when cooled. Agar is poured hot into petri dishes, slanted tubes or agar stab tubes 43 The preparation of Media There is a wide variety of media which are used in microbiology, but the procedures used in their preparation are generally the same. Media can be solid, semi solid or liquid 44 Media can be solid, semi solid or liquid 45 Equipment needed: Large beaker ( 1000 ml or larger) Balance Thermometer ( -10°C to 110°C) Heat source, hot pads 1000 ml bottle with a cap Funnel autoclave 46 How to prepare MacConkey Media ( e coli & other gm- bacteria Weigh out the following and dissolve in 1000 ml of distilled water: ◦ Peptone 17.0 g ◦ Proteose peptone 3.0 g ◦ Lactose 10.0 g ◦ Bile salts no 3 1.5 g ◦ NaCl 5.0 g ◦ Agar 13.5 g ◦ Neutral red 0.03 g ◦ Crystal violet 0.001 g 47 Dehydrated Media 48 Procedure for preparing media Weigh out the dehydrated media Dissolve in distilled water Sterilizing it ( usually in an autoclave) Pouring the plates Pre incubation to check for contamination, QC and to dry out the plates PH media Storage 49 Procedure for preparing media Storage Most media is stored in the fridge ( 2- 8 degrees C) Also, all media must have labels on them. The label must include what type of media it is, lot number and expiry date. 50 QUALITY CONTROL TESTS ON PREPARED MEDIA 1. pH value: check that the pH of the prepared medium, when tested in final form at ambient temperature (25°C) lies within the range given on the product label. The medium should be discarded if the pH value lies outside the specified range. 51 QUALITY CONTROL TESTS ON PREPARED MEDIA 2 Sterility: a representative sample of each lot/batch of medium should be incubated for 2-5 days at 35-37°C and at room temperature. There should be no evidence of microbial growth after incubation. Discard all sterility samples when the tests have been completed. 52 QUALITY CONTROL TESTS ON PREPARED MEDIA 3 Growth performance: test the growth support properties of the product by inoculating the medium with appropriate stock cultures and/or fresh isolates. Use a standard inoculation procedure and examine the quantitative and qualitative results obtained. 53 QUALITY CONTROL TESTS ON PREPARED MEDIA 4 Stability: periodically perform the above procedures on stored prepared media in order to determine whether the storage conditions will give optimal results. 54 Labelling Media Before you store media, the media must be labelled with the following; The type of media Lot number Expiry date 55 56 Categories of Media Supportive/ Basal Enrichment Selective Differential 57 Supportive / Basal media Supplies minimum requirements for growth E.g. Mueller Hinton ( MH) 58 Supportive Media 59 Enrichment media Inhibitsthe growth of normal flora and enhances the growth of pathogens Examples of enrichment media ◦ Chocolate agar ◦ Blood agar ( 5% sheep’s blood) 60 Chocolate agar plate Blood agar plate Enrichment Agar 61 Selective Media Inhibitsthe growth of some organisms while enhancing the growth of others Used for sputum and stool cultures Examples of selective media: ◦ Salmonella – Shigella (SS) ◦ Mannitol salt agar – for staph aureus ◦ Lim Broth – vaginal/anorectal swabs for Group B strep ( all pregnancies are screened for gr B strep) ◦ MacConkey’s 62 E. coli Staphylococcus Staph aureus Selective Media 63 Differential Media Have indicators that some bacteria will use ( pink) and others won’t.( colorless) Examples of differential media: ◦ MacConkey’s - used in most cultures 64 Differential Media MacConkeys Agar 65 The most common media used in a microbiology lab is: ◦ Blood ◦ MacConkeys ◦ Chocolate 66 Order of Inoculation  Supportive  Enrichment  Differential  Selective 67 Setting up cultures Culture plates can be inoculated from swabs or other sources. If a specimen comes without a swab( e.g. urine use a sterilized inoculation loop.) or a sterile swab for fluids If a swab or other source is used, roll the swab or other source across the media to inoculate. Inoculation – the original introduction of microorganisms onto a culture plate 68 Streaking plates Bacteria frequently occur in mixed populations. It is very rare to find a single occurring species of bacteria.  To study the cultural, morphological, and physiological characteristics of a bacteria that might be causing the infection ( pathogen), we have to isolate from the other bacteria. in a clinical sample. When the bacteria is streaked and isolated, the causative agent of a bacterial disease can be identified. 69 Streaking plates The three-phase streaking pattern, known as the T-Streak. 70 Step 1 A sterile loop or sterile swab dipped into an inoculum such as a broth or patient specimen. The inoculation loop( or swab)is then dragged across the surface of the agar back and forth in a zigzag motion until approximately 30% of the plate has been covered. 71 Step 2  The loop then is re-sterilized and the plate is turned 90 degrees. Starting in the previously streaked section, the loop is dragged through it two to three times continuing the zigzag pattern. 72 Step 3 The procedure is then repeated once more being cautious to not touch the previously streaked sectors. 73 Conclusion Each time the loop gathers fewer and fewer bacteria until it gathers just single bacterial cells that can grow into a colony. The plate should show the heaviest growth in the first section. The second section will have less growth and a few isolated colonies, while the final section will have the least amount of growth and many isolated colonies. 74 Streaking Diagram 75 76 Autoplak 77 Autoplak 78 Bacterial Physiology Aerobic bacteria requires 21% oxygen Anaerobic bacteria grow in the absence of oxygen Facultative anaerobes can grow in the presence or absence of oxygen 79 Bacterial Physiology Microaerophilic must have 5 % oxygen ( example : Campylobacter) Capnophilic grows in the presence of 5 to 10% CO2 ( Example: Neisseria gonorrhea ) 80 Bacterial Physiology 80% of bacteria is H2O – therefore moisture is required for growth. 70% humidity is required in the incubator Most bacteria that affects humans require an isotonic environment ( 0.9% saline) PH 7.2 – 7.6 Temperature 35 - 37° 81 Anaerobic organisms Put in an anaerobic jar with a catalyst. Break the catalyst pouch. Pour water inside to activate the catalyst which depletes O2 from the jar Use an indicator – Methylene Blue  - aerobic – blue color  - anaerobic – white color 82 Anaerobic Jar 83 Microaerophilic Jar For organisms that need 5 % oxygen Setting this environment is the same as setting the anaerobic jar. Only difference is the pouch. This pouch creates a 5 % oxygen environment Examples. Campylobacter 84 Microaerophilic Jar 85 Candle Jar 86 Carbon Dioxide enriched environment ( 5 – 10% CO2) Place the specimen in a jar Light a candle and close the lid Candle will burn out and enrich the atmosphere in the jar with CO2 Example – Neisseria GC 87 Growth Curve Lag Phase – adjustment period to a new medium and environment. The bacteria is preparing for growth. ( 8 hours) Log phase – very rapid growth period for bacteria. The number of bacteria doubles in proportion to time. ( 36 hours) 88 Growth Curve Stationary phase – nutrients are used up. There is now a balance between cell growth and cell death. ( a few hours) Death phase – bacteria stop multiplying 89 Culture plates are incubated for 18-24 hours before colonies appear. Most plates are incubated no longer that 48 hrs. 90 DIFFERENT BODY SITES AND WHAT T0 INOCULATE Before you inoculate plates, look to see where the specimen come from, what type of specimen , and the priority. Example ; surgical specimens should be inoculated first. Sterile fluids have priority. Stat specimens are always first 91 DIFFERENT BODY SITES AND WHAT T0 INOCULATE Throat swabs - BA plate - looking for Group A Strep 92  Urine - BA plate - MacConkey’s plate You have to use a calibrated look ( 0.001 ml or 1 ul) Quantitative urine cultures( you count the colonies) Technical urine is sterile. The most organisms that cause UTI are organisms from the Enterobacteriaceae family. Example E.coli. There is also Staphylococcus saprophyticus 93  Vaginal swabs( Pregnancy) - LIM broth , then after 24 hr incubation, plate to a BA plate Looking for Group B Strep 94  Ear swabs – CA plate - BA plate - MacConkey’s plate Looking for Streptococcus pneumoniae, Haemophilus influenza, Moraxella catarrhalis, Pseudomonas aeruginosa 95  Eye Swabs – CA plate - BA plate Conjunctivitis or pink eye Looking for Haemophilus influenzae, Streptococcus pneumoniae 96 Urethral swabs – Thayer Martin Plate Looking for Neisseria gonorrhoeae. Alsocervix swabs for GC Vaginal swabs not acceptable for GC 97 Sputum specimens – CA, BA, and Mac Looking for Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, and Klebsiella species. 98 Note 1: Gram stain may be used to determine the acceptability fof the sputum. If the gram stain shows a lot of pus cells ( > 25/LPF) then the specimen is accepted. If it show a lot of epithelial cells , then the specimen is rejected Note 2: Anaerobic cultures are not acceptable in most respiratory specimens 99 CSF – BA - CA For Meningitis Looking for Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae CSF ARE ALWAYS DONE STAT 100  Wound Swabs – CA plate - BA plate - MacConkey’s plate - Anaerobic media when necessary. 101 Looking for Staphylococcus aureus, Group A Streptococci, Pasteurella multocida, Enterobacteriaceae family, anaerobic bacteria, and other types of microorganisms, like yeast. Anaerobes examples: Clostridium sp, Propionibacterium, Bifidobacterium 102  Fluids CA plate - BA plate - CA - Anaerobic media when necessary. - Pediatric bottles 103 Fluids For a true joint fluid (not a "bursa" fluid), we plant it to a blood, chocolate and anaerobic blood (BAK) agar. We also inoculate 'some' (0.5mls to 1.0 ml depending on how much fluid is received) into a PEDS bottle (yellow top blood culture bottle), which gets loaded onto the BacT and incubated for 5 days. If the fluid is non-viscous, we would 104 Fluids If the fluid is non-viscous, we would make a cytospin gram and centrifuge the sample. However, joint fluids are usually always viscous so we make a gram stain straight from the original sample and directly plant the sample to the agar. If they request anaerobes we also 105 Looking for Staphylococcus aureus, Group A Streptococci, Enterobacteriaceae family, anaerobic bacteria, and other types of microorganisms, like yeast. Anaerobes examples: Clostridium sp, Propionibacterium, Bifidobacterium Bacteroides, Fusobacterium, 106  Stool – BA plate, - MacConkey’s, - HE - Hektoen enteric agar or XLD- Xylose-Lysine- Desoxycholate or SS Agar ( Salmonella/ Shigella) - Campylobacter Blood Agar - MacConkey Sorbital Agar CIN (Cefsulodin-Irgasan-Novobiocin) 107  Looking for Salmonella species, Shigella species, Campylobacter species, E. coli O157:H7, Yersinia species. Vibrio species – there has to be a special request. Media is TCBS Agar - (Thiosulfate Citrate Bile Salts Sucrose) 108 Note : Campylobacter must be inoculated on Skirrow's media. Incubated at 42 degrees C Must be in a microaerophilic jar ( 5 % O2) 109 Blood cultures General  Determine presence & extent of infection  Identify type of organism responsible & best antibiotic to use  Ordered because: 1. Patient has a condition in which bloodstream invasion is possible(Suspected bacteremia) 2. Fever of unknown origin (FUO) 110 Blood culture continued Bare collected with the butterfly method. A blood culture adapter is used Using the butterfly to draw blood for a blood culture, fill the aerobic bottle first. This will prevent the air from the tubing of the butterfly to go into the anaerobic bottle. 111 Blood culture continued All patients weighing more than 27 kg (approximately >9 years of age) must have 40 mL of blood drawn from two different venipunctures or sites within one hour. The two sites should always be sampled at the same time (within 1 hour) and all specimens should 112 Blood culture continued Each site must collect 20 mL of blood equally divided into one aerobic (blue) and one anaerobic (purple) bottle. Failure to collect sufficient volume from two different collections significantly impairs diagnosis and may lead to false- negative or false-positive results. 113 Blood culture continued 114 Blood culture continued Do not place more than 12 mL of blood in a single blood culture bottle. While lower volume collection in the bottle is not detrimental, the total collection of blood for a blood culture should not be less than 30 mL. 115 Collection volumes for pediatric patients