Successfully reported this slideshow.
We use your LinkedIn profile and activity data to personalize ads and to show you more relevant ads. You can change your ad preferences anytime.

Microbiological culture sensitivity test

7,123 views

Published on

Microbiological culture sensitivity test.
antimicrobial sensitivity test

Published in: Education
  • D0WNL0AD FULL ▶ ▶ ▶ ▶ http://1lite.top/wKhGz ◀ ◀ ◀ ◀
       Reply 
    Are you sure you want to  Yes  No
    Your message goes here
  • There is a useful site for you that will help you to write a perfect and valuable essay and so on. Check out, please ⇒ www.WritePaper.info ⇐
       Reply 
    Are you sure you want to  Yes  No
    Your message goes here
  • If you’re struggling with your assignments like me, check out ⇒ www.HelpWriting.net ⇐. My friend sent me a link to to tis site. This awesome company. After I was continuously complaining to my family and friends about the ordeals of student life. They wrote my entire research paper for me, and it turned out brilliantly. I highly recommend this service to anyone in my shoes. ⇒ www.HelpWriting.net ⇐.
       Reply 
    Are you sure you want to  Yes  No
    Your message goes here
  • This information is rather useful smile emoticon Thanks smile emoticon By the way, HelpWriting.net provides efficient writing services smile emoticon
       Reply 
    Are you sure you want to  Yes  No
    Your message goes here
  • D0WNL0AD FULL ▶ ▶ ▶ ▶ http://1lite.top/wKhGz ◀ ◀ ◀ ◀
       Reply 
    Are you sure you want to  Yes  No
    Your message goes here

Microbiological culture sensitivity test

  1. 1. Microbiological Culture Sensitivity Test. Antibiotic Sensitivity Test ( AST ) Akhil Joseph Pharm.D IVth Year. Reg.No: 13Q0402
  2. 2.  According to the new ISO 20776-1 standard, which is valid all over the world, these terms are defined as follows:  Susceptible (s): A bacterial strain is said to be susceptible to a given antibiotic when it is inhibited in vitro by a concentration of this drug that is associated with a high likelihood of therapeutic success.  Intermediate (i): The sensitivity of a bacterial strain to a given antibiotic is said to be intermediate when it is inhibited in vitro by a concentration of this drug that is associated with an uncertain therapeutic effect.  Resistant (r): A bacterial strain is said to be resistant to a given antibiotic when it is inhibited in vitro by a concentration of this drug that is associated with a high likelihood of therapeutic failure.
  3. 3. mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L Ampicillin 0.25 0.5 1 2 4 8 16 32 Ampicillin/sulb actam 0.25 0.5 1 2 4 8 16 32 Piperacillin/taz obactam 0.5/2 1 2 4 8 16 32 64 Cefuroxime 0.125 0.25 0.5 1 2 4 8 16 Cefotaxime 0.125 0.25 0.5 1 2 4 8 16 Imipenem 0.125 0.25 0.5 1 2 4 8 16 Gentamicin 0.125 0.25 0.5 1 2 4 8 16 Doxycyclin 0.125 0.25 0.5 1 2 4 8 GC Cotrimoxazole 1 2 4 8 16 32 64 128 Ciprofloxacin 0.03 0.06 0.125 0.25 0.5 1 2 4 Levofloxacin 0.03 0.06 0.125 0.25 0.5 1 2 4 Moxifloxacin 0.03 0.06 0.125 0.25 0.5 1 2 4 Schematic representation of a microtitration plate for the determination of minimal inhibitory concentrations (MICs)
  4. 4.  The results for a particular tested strain of E. coli.  The MIC values for this strain and the sensitivity ratings that will be assigned to them henceforward by EUCAST (The European Committee on Antimicrobial Susceptibility Testing) , are:  ampicillin = 4 mg/L (i); ampicillin/sulbactam = 1 mg/L (i); piperacilin/tazobactam = 2 mg/L (s); cefuroxime = 4 mg/L (i); cefotaxime = 0.125 mg/L (s); imipenem = 0.5 mg/L (s); gentamicin = 0.25 mg/L (s); doxycycline = 8 mg/L (Ø); cotrimoxazole >128 mg/L (r); ciprofloxacin <0.03 mg/L (s); levofloxacin <0.03 mg/L (s); moxifloxacin = 0.06 mg/L (s);  GC, growth control; Ø, no data, because the combination of organism and antibiotic is unsuitable; i, intermediate; s, susceptible; r, resistant.
  5. 5. INTRODUCTION  Antimicrobial susceptibility testing (AST) is indicated for pathogens contributing to an infectious process that warrants antimicrobial therapy if susceptibility to antimicrobials cannot be predicted reliably based on knowledge of their identity.  Some organisms have predictable susceptibility to antimicrobial agents (ie, Streptococcus pyogenes to penicillin), and empirical therapy for these organisms is typically used. Therefore, AST for such pathogens is seldom required or performed. In addition, AST is valuable in evaluating the activity of new and experimental compounds and investigating the epidemiology of antimicrobial resistant pathogens.
  6. 6.  Once we have identified the bacterium which is causing the infection we need to find out the antibiotics that would be effective against it. This is done by antibiotic sensitivity testing.  Antibiotic sensitivity or microbiological culture sensitivity are the in-vitro procedures to determine the susceptibility of bacteria to antibiotics.  Because susceptibility can vary even within a species (with some strains being more resistant than others), antibiotic susceptibility testing (AST) is usually carried out to determine which antibiotic will be most successful in treating a bacterial infection in vivo.
  7. 7.  The same can be used to study the emergence of antibiotic resistance and spread of resistant organism in a population.  Antibiotic sensitivity testing will control the use of Antibiotics in clinical use.  Testing will assist the clinicians in the choice of drugs for the treatment of infections.
  8. 8. Why is sensitivity analysis done ?  Usually almost all the bacteria in infectious disease are drug resistant.  Unfortunately, nowadays many bacteria are resistant to common antibiotics.  Thus sensitivity tests are better tool to quickly determine if bacteria are resistant to certain drugs.  Hence sensitivity test is performed to select the correct antimicrobial drug of choice, hence cost effective and rational therapy can be provided.  Lower the risk of emergence of antibiotic resistance.  It may also help to identify the pathogen.
  9. 9. Limitations o It helps us to measure only the antimicrobial activity against a bacteria under laboratory conditions and not in the patients. o The patients clinical condition, type and site of infection, drug hypersensitivity, ADME, characters of the patients are not taken in to consideration in sensitivity testing techniques.
  10. 10. Culturing and Sensitivity Testing Specimen Collection. Samples must be collected and handled properly to obtain reliable results. Poor collection techniques may result in lack of bacterial growth or abundant growth of bacterial contaminats. Sample shoud be collected before the antibiotic therapy to assure the best growth of the pathogen. If antibiotic therapy is already initiated, then the sample must be collected before the next dose.
  11. 11. Identifying Bacteria  When a sample is submitted to laboratory for bacterial culture and antibiotic sensitivity, the clinician should include the information like the site of sample collection and type of lesion. This help the microbiologist to choose which nutrient media and growth conditions to be used.  Samples of bacterial culture are applied to plates of various growth media with a sterile loop, effectively spreading the bacterial organism over the surface of each plate in a single layer.  Once inoculated the plates are incubated in an environment with controlled temperature, humidity, oxygen and carbon dioxide levels are optimum for bacterial replication.  Each bacterial organism grows into a cluster called a colony, and individual colonies are inoculated onto new separate media, creating pure samples.  Identification of the cultured bacteria is based on the characteristics of colony growth and appearance as well as biochemical testing of the individual colonies.
  12. 12. Antibiotic Sensitivity Testing.  Once identified the bacteria undergoes testing to identify the antibiotic most likely to inhibit their growth.  For this purpose generally two methods are used, 1) Disk Diffusion techniques. 2) Broth Dilution techniques.
  13. 13. Disk Diffusion techniques. Agar Disk Diffusion Testing  In many clinical microbiology laboratories an agar disk diffusion method is routinely used for the testing of common, rapidly growing, and some fastidious bacterial pathogens, allowing categorization of most such isolates as susceptible, intermediate, or resistant to a wide range of antimicrobial agents.  A disc of blotting paper is impregnated with a known volume and appropriate concentration of antimicrobial placed on a plate of sensitivity agar inoculated with test organism.
  14. 14.  The antimicrobial agents then diffuse from the disks through the agar, and as the distance from the disks increases, the drug concentrations decrease in a logarithmic fashion, creating gradients of drug concentrations in the medium around the disks.  Simultaneously with the diffusion of the drugs, the bacteria inoculated to the agar surface not inhibited by the concentrations of the antibiotics in the agar multiply, creating a visible lawn of growth. In areas where the test organism is inhibited by the antimicrobial agents, growth fails to occur, resulting in zones of inhibition around each active drug. The inhibitory zone diameters are influenced by the diffusion rates of the various antimicrobial agents through the agar  After 24 hours, the culture is examined for areas of growth around the disc.
  15. 15.  The zone sizes are inversely proportional to the logarithms of the antibiotic MICs. After incubation at recommended temperatures, atmospheric conditions, and times, depending on the pathogen under study, the diameters of the zones of inhibition are measured in millimeters and compared to a standard table of predetermined zone widths representing antibiotic concentrations in the agar that correlates with that of the antibiotic concentration achievable in the plasma of a patient using the manufacturers recommended dosage.  If the zone of inhibition is wider than the pre determined zone, the bacterial species is considered to be susceptible(S) to the antibiotic. If bacteria grows within the pre determined zone width the species is considered as resistant (R). An intermediate (I) designation is used if the zone of inhibition approximates the predetermined zone width.  Growth for sensitive strains are inhibited for a distance while for resistant strains it grows up to the edge of the disc.  The volume, moisture content, PH, constituent of agar medium, concentration, storage and application of dose influence the diffusion technique.  Agar diffusion sensitivity tests are carried out either by Kirby-Bawer (KB) method, ICS method or by Stocks method.  Modified KB method is recommended by the National Committee for clinical Laboratory Standards (NCCLS) and the WHO.
  16. 16. ADVANTAGES  (1) it is technically easy to perform and results are reproducible,  (2) the reagents and supplies are inexpensive,  (3) it does not require the use of expensive equipment,  (4) it generates categorical interpretive results well understood by clinicians, and  (5) it allows for considerable flexibility in the selection of antibiotics for testing. DISADVANTAGES  The exact conc. of antibiotic that inhibited the bacterial growth is not known.
  17. 17. Broth Dilution techniques.  Dilution sensitivity tests usually measures the minimum inhibitory concentration (MIC) or minimum bactericidal concentration (MBC) required to kill the bacteria.  Test tubes or wells containing increasing concentrations of each antibiotic to be tested, from 0.0312 to 512mcg/ml, are inoculated with a fixed volume of nutrient broth containing a standard concentration of bacteria is added.  The conc. Of antibiotic in each tube is double as that of previous tube.  After overnight incubation the tubes are checked for turbidity, turbidity indicates growth of bacteria, the lowest antimicrobial required to prevent visible growth is taken in to consideration.  MIC is the lowest concentration of antibiotic that inhibits the bacterial growth.
  18. 18.  Dilution technique needs,  Careful standardization  Broth and agar medium  Antimicrobial solution  Incubation time and  Dilution time
  19. 19. Ideally, clinicians should always choose a drug to which the identified bacteria are considered susceptible and should avoid agents to which they are intermediate or resistant.
  20. 20. General requirements for sensitivity testing. 1. Sensitivity testing agar. Suitable media include Mueller Hinton agar, Iso sensitest agar and Gibco sensitivity testing agar no.2. Mueller Hinton agar(MHA) Composition  Meat infusion 2.0 g/l  Casein hydrolysate 17.5 g/l  Starch 1.5 g/l  Agar-agar 13.0 g/l
  21. 21. 2.ANTIMICROBIAL DISC  This disc should be refrigerated at a temperature instructed by the manufacturer.  This should not be used after expiry date.  The working stock disc should be warmed to room temperature, avoid keeping in direct sunlight.
  22. 22. ANTIMICROBIAL RESISTANCE  Antimicrobial resistance can arise in bacteria in several ways.  Microbes acquire resistance after a change in their DNA.  Such changes may occur by  genetic mutation i.e. by alteration in the structure of their own DNA. • genetic exchange i.e. by acquisition of extra- chromosomal DNA from other bacteria.
  23. 23.  Genetic exchange is the most common cause of serious clinical drug resistance because it can produce resistance to multiple drugs.  In genetic exchange , the resistance genes are transferred from one bacterial species to another by means of discrete, movable, extra chromosomal DNA elements called TRANSPOSONS.
  24. 24.  Transfer of transposons between bacteria can occur by • Conjugation i.e, direct physical mating between bacteria. • Transduction i.e, through the agency of bacteriophages. • Transposition i.e, by means of plasmids which are transferable, extra chromosomal DNA molecule.
  25. 25. DRUG RESISTANCE It refers to unresponsiveness of a micro- organism to an antimicrobial agent. They are of 3 types: i. Natural resistance ii. Acquired resistance iii. Cross resistance.
  26. 26. NATURAL RESISTANCE  Some microbes have always been resistant to certain AMA.  They lack the metabolic process or the target site which is affected by the particular drug.  eg. gram negative bacilli are normally unaffected by Pencillin G  M.tuberculosis is insensitive to tetracyclines.
  27. 27. ACQUIRED RESISTANCE  It is the development of resistance by an organism (which was sensitive before) due to the use of an AMA over a period of time.  Eg; methicillin resistant Staphylococcus aureus.
  28. 28. CROSS RESISTANCE  Cross-resistance is the tolerance to a usually toxic substance as a result of exposure to a similarly acting substance.  It is a phenomenon affecting e.g. pesticides and antibiotics as an example.

×