Professional Documents
Culture Documents
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Bacteriology Laboratory
Bacteriology Laboratory makes the Backbone of any Hospital and without which no hospital can function to the Minimal needs, All the Microbiologists and Lab Dr.T.V.Rao MD professionals need
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Microorganisms
Microbiology and the Role of the Medical Technicians Medical technicians can be
n
Assists physician / Microbiologists Obtains specimens Prepares specimens for direct examination Prepares specimens for transportation to reference laboratory If office has a POL, performs
Classification by structure
Subcellular DNA or RNA surrounded by a protein coat viruses Prokaryotic simple cell structure with no nucleus or organelles bacteria Eukaryotic complex cell 10/14/12 Dr.T.V.Rao MD 66
Bacteria:
Special groups
Chlamydia
Mycobacteria bacilli with a cell wall that differs from most bacteria Rickettsia
Cell wall structure differs from other bacteria Live and grow within other living cells
Very small
o o o
Bacteria
Shape Ability to retain dyes Ability to grow with / without air Biochemical reactions
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Bacteria:
Identification (cont.)
Classification and
Shape
Bacteria:
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Morphological characteristics Biochemical characteristics Signs and symptoms they cause in the host they are infecting Virulence factors Pathophysiology of infection
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diagnosis
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Culture specimen
n Culture
medium contains
nutrients
n Examine
Different media are used to culture microorganisms, be certain that you are using the appropriate media for your organism. Always use sterile technique to prevent contamination.
Choose the type of media (liquid or plate) appropriate for your investigation or application.
Sterile liquid culture tubes and media plates can be prepared in advance and stored in the 10/14/12 Dr.T.V.Rao MD refrigerator for later use (2 weeks
Liquid culture tubes, solid slant tubes, and petri plates can be used to culture microbes. Media and lab materials should be sterilized prior to use; an autoclave or a pressure cooker can be used in the sterilization process.
Serial dilution and plate count techniques are used to estimate microbial 10/14/12 Dr.T.V.Rao MD
Specimen Collection
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Specimen Collection
(cont.)
Devices
Use appropriate collection device or specimen container Sterile swabs absorbent material on the tip
Specimen Collection:
Guidelines
o Avoid causing harm, discomfort, or undue o embarrassment o Collect from appropriate site o Obtain specimen o at correct time 10/14/12 Dr.T.V.Rao MD o Use appropriate o
Obtain sufficient quantity of specimen Obtain specimen prior to the start of antimicrobial therapy Label correctly
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Swab back of throat in the area of the tonsils Avoid touching any structures in the mouth Prepare culture plate or prepare correctly
Specimen Collection
o o
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Nacldissolves keratin that can mask KOH mixed with specimen of fungus slide presence of a glass n Presence of pathogen and movement of microorganism MD 10/14/12 Dr.T.V.Rao 2323
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o o
Preparation and Examination of Stained Specimens Grams tentative diagnosis Quick, stain
Moderate- complexity test Differentiation between types of infections Bacteria either retain or lose purple
color
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Using aseptic technique remove a colony from a plate or cells from your slant. Be carefully to gently touch the surface of your culture with the inoculating loop. Make a circular motion in the middle of the circle to spread the cells equally in this region of the slide Add a drop of water in the middle
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Mix again
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Making a Smear
Wash the glass slide thoroughly with soap and water then rinse with 95% alcohol to sterilize. 2. Allow the slide to dry properly. 3. Pass the clean slide over a flame with its face down to further sterilize it. (Make sure to hold it by its edge)
4. Draw a small circle on the slide so you can put your bacteria on the back of the marked area.
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Smear Preparation
Smear Preparation Only a small amount of bacterial culture should be used. Thick smear causes overcrowding of a large number of cells. Two different media require two different techniques Liquid Medium/ Broth Culture 1.
Dr.T.V.Rao MD 2727 Take the loop and hold it in the
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Run the slide through the flame until the slide is warm ( The frosted side should be down) This fixes the bacteria to the slide Let the slide cool Place in the metal tray or in the rack
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Media Types
General
Purpose Media Enriched Media Selective Media Differential 10/14/12 Dr.T.V.Rao MD Media
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Culturing Microorganisms
Liquid culture: bacteria, algae, and some fungi can be reared in culture tubes (test tubes) in a liquid medium.
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Liquid medium is best when you Dr.T.V.Rao MD 3030 want to rapidly increase the
Culturing Microorganisms
Culture Plates: Liquid medium is solidified using agar (Agarose) and poured as a thin layer in the bottom of a culture dish (also sometimes called petri plate)
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Culture plates are used when you want to test (1) antibiotic sensitivity, (2) estimate culture concentrations from environmental samples, or (3) isolate Dr.T.V.Rao MD 3131 individual colonies from environmental
Medium nutrients Place in incubator for growth colony develops as microorganism multiplies
Sterile Technique
When culturing bacteria or other microorganisms, it is important to keep your work area as clean as possible. This prevents the introduction of other microorganisms from the environment into your culture.
The techniques used to prevent contamination are referred to as 10/14/12 Dr.T.V.Rao MD 3333 sterile techniques.
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Sterile Technique
1.
Start by washing your down your work or lab benches with a surface disinfectant. The most commonly used disinfectants for lab use are:
1.
2.
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Aseptic Technique
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Sterile Technique
1.
Start by washing your down your work or lab benches with a surface disinfectant. The most commonly used disinfectants for lab use are:
1.
Culture media
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Media Types
General Purpose Media: Supports the growth of many microorganisms i.e. Nutrient agar Enriched Media: Has special nutrients to encourage the growth of fastidious heterotrophs i.e. Blood Agar Selective Media: Favors the growth of one type of microorganisms and inhibits the growth of others Luria + penicillin Agar Differential Media: Distinguishes between different groups of bacteria on the basis of biochemical characteristics i.e. Eosin Methylene Blue Agar
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Clean and surface sterilize your work area as detailed in the section on Sterile Technique. Use either disposable inoculation loops or a metal loop that can be heat sterilized to inoculate plates, slants, and liquid culture tubes.
If using a metal loop, be sure to cool the loop by touching the sterile cooled liquid media or the sterile culture plate before the placing the 10/14/12 in yourDr.T.V.Rao MD 4141 loop live culture. Failure to
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Culturing Specimens
(cont.)
Transfer some of the specimen onto a culture plate Label the plate correctly Qualitative analysis determination of type of pathogen Quantitative analysis number of bacteria present in sample
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1. Wrap fingers of non dominant hand around the culture tube containing broth for transfer 2. Using the pinkie finger of your dominant hand twist the red cap from the tube. Hold in your pinkie and do not place it on the counter 3. Pass the mouth of the culture tube across the flame 4. Direct the inoculating needle into 10/14/12broth. Dr.T.V.Rao MD 4747 the
Procedure for Transferring Microorganisms to a Slant 7. Twist off the red cap
8. Flame the mouth of the slant tube 9. Direct the inoculating needle into the tube and stab the agar in the base( butt) 10. Withdraw on the entry line and when you reach the surface make a simple streak along the face. 10/14/12 Dr.T.V.Rao MD 4848
Culturing Specimens
(cont.)
Transfer some of the specimen onto a culture plate Label the plate correctly Qualitative analysis determination of type of pathogen Quantitative analysis number of bacteria present in sample
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Colony Morphology
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Sensitive no growth n
Filter paper Intermediate little growth containing Resistant overgrown antimicrobial agents placed on inoculated agar plate
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Microorganism Categories
By genetics to show how they are related By tissues they infect to show how they cause disease
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Biosafety Level 1
Prohibit eating, drinking and smoking in the laboratory Pipetting by mouth strictly forbidden
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Biosafety Level 1
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Keep work areas uncluttered and clean No food in lab refrigerator Minimize splashes and aerosols Decontaminate work surfaces daily
Decontamination
Sterilization Disinfection
Decontamination
Types
Chemical
Decontamination
Chemical
Hypochlorite Solutions
Large Spills/Large Organic Load
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In case of a spill
Wear disposable gloves Cover large blood spill with paper towels and soak with 1% (10000 ppm) of household bleach and allow to stand for at least 5 minutes Small spill - wipe with paper towel soaked in 1% bleach Discard contaminated towels in infective waste containers
Wipe down the area with clean towels soaked in a 10/14/12 same dilution of household bleach Dr.T.V.Rao MD 6464
doctortvrao@gmail.com
Dr.T.V.Rao MD 6565
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