Medical Lab Microbiology Practical-22025.pptx

Microbiology Practical: Culture media preparation in a medical lab

1. Microbiology Practical 2 CULTURE MEDIA PREPARATION I nstructors : Chelangat Abarteneko , Amadile Lawrence & Edema Wiliam

2. Objectives By the end of the session, you will be able to : Know Microbiology equipment Define culture media Explain why it is important to prepare it Classify and identify the different types of common culture media and their use Prepare different culture and identification media Conduct quality control checks on prepared media Properly store and dispose prepared culture Media

3. Microbiology equipment Basic clinical Microbiology laboratory: BSC-2 Autoclave Fridge Freezer Weighing balance Wire loops Gas cylinder/candle/Bunsen burner Calibrated ruler Microscope 0.5Macfarland standard Incubator(Aerobic) Anaerobic jar Water bath Advanced lab Automated blood culture machine BD 9050/FX40/FX200 BACTALERT Automated ID &AST machine MALDITOF/VITEC2 Compact(MS) API BSC-3 Automated Gram stain machine PH meter Densio meter CO2 incubator/Anerobic incubator

4. Introduction In natural environments, microorganisms usually exist as mixed populations. However, if we are to study, characterize, and identify microorganisms, we must have the organisms in the form of a pure culture. Apure culture is one in which all organisms are descendants of the same organism. Techniques for obtaining pure cultures from a mixed population will be studied in this Lab subsequently Bacteria have to be grown (cultured) for them to be identified.  By appropriate procedures they have to be grown separately (isolated) on culture media and obtained as pure for study. History  The original media used by Louis Pasteur urine or meat broth  Liquid medium  Solid medium –– diffuse growth discrete colonies

5. Introduction Bacteria have to be grown (cultured) for them to be identified. By appropriate procedures they have to be grown separately (isolated) on culture media and obtained as pure for study History The original media used by Louis Pasteur urine or meat broth Liquid medium Solid medium –– diffuse growth discrete colonies

6. 1. Based on consistence Solid media Liquid media Semi solid medium Classification 2. Classification based on purpose Enriched media Enrichment media Selective media Differential media Transport media Indicator media 3. Based on Oxygen requirement Aerobic media Anaerobic media

7. 2. Classification based on purpose i ). SIMPLE MEDIA Eg : Nutrient Broth, Nutrient Agar - NB consists of peptone, meat extract, NaCl, NB + 2% agar = Nutrient agar ii). ENRICHED MEDIA Classification. Cont . Chocolate Agar Most common in routine diagnostic labs: Eg : Nutrient Broth, Nutrient Agar NB consists of peptone, meat extract, NaCl NB+0.5 Glucos e= Glucose Broth NB + 2% agar = Nutrient agar Agar conc. reduced to (0.2 media 0.5%)= Semi solid Enriched media: Substances like blood, serum, egg are added to the basal medium Used to grow bacteria that are exacting in their nutritional needs ( fastidious organisms E.g.: Blood agar, Chocolate agar

8. iv) ENRICHMENT MEDIA Liquid media used to isolate pathogens from a mixed culture. Media inhibitory substances to suppress the unwanted organism. Eg: Selenite F Broth – for the isolation of Salmonella, Shigella Alkaline Peptone Water – for Vibrio cholerae Classification. Cont … Alkaline peptone wate Tetrathionate broth

9. iv). SELECTIVE MEDIA The inhibitory substance is added to a solid media. Eg : Mac Conkey’s medium for gram negative bacteria TCBS – for V. cholerae LJ medium – M. tuberculosis Wilson and Blair medium – S. typhi Potassium tellurite medium – Diphtheria bacilli Xylosse lysine Deoxycholate medium(XLD)- salam / sh Mac Conkey’s TCBS Classification Cont.

10. Selective media Cont.. Potassium Tellurite media LJ media

11. v). INDICATOR MEDIA/Differential These media contain an indicator that changes its colour when a bacterium grows in them. Eg : Blood agar Mac Conkey’s medium Christensen’s urease medium Urease medium Classification. Cont … Haemolysis on Blood agar

12. Indicator media Cont… Blood agar (BA): Erythrocytes are incorporated into nutrient agar medium Certain bacteria produce products that lyse Red Blood Cells Alpha-hemolytic- partial lysis Beta-hemolytic- complete lysis Gamma-hemolytic- no lysis

13. Indicator media Cont… Urease medium Urease neg (left), urease post(Right) pink

14. vii) ANAEROBIC MEDIA These media are used to grow anaerobic organisms. Eg: Robertson’s cooked meat medium, Thioglycolate medium. Classification. Cont … RCMM THIOGLYCOLATE MEDIUM

15. Anaerobic media Cont … § § Eg :

16. v). TRANSPORT MEDIA Media used for transporting the samples. Delicate organisms may not survive the time taken for transporting the specimen without a transport media. Eg: Stuart’s medium – non nutrient soft agar gel containing a reducing agent Buffered glycerol saline – enteric bacilli Classification. Cont …

17. MSA Cont…

18. Common media used Mueller Hinton Agar: Rich medium that support the growth of most microorganism. It is commonly used for antibiotic susceptibility testing: o disk diffusion antibiotic susceptibility; o antibiotic serum level measurements; o MBC determination

19. …protective clothing …hand washing …bench cleaning Aseptic Techniques For Culture media Preparation Dos for media preparation Avoid talking while pouring media Ensure clean benches Use gloves Do not multi task e.g. sample processing and media preparation Ensure you have a functional autoclave Ensure you have a functional weighing balance Prepare media in sterile room(media room) Observe other safety rules

20. Media preparation Cont.. Aseptic techniques in media preparation

21. Media preparation When lab personnel make media, they measure out a quantity of dry powdered nutrient media, add water and check the pH. Measurements are done according t manufacturer’s recommendation The media is then dispensed into bottles (flask, tube), capped and autoclaved at 121°C (15 psi) for 20 minutes. Once the media is autoclaved it is sterile (all microoranism forms killed)

22. Preparation of 5% BA Prepare 200mls Use the formula RV/O=X R=Required concentration=5% V=Required volume=200mls O=Original conc of blood=100% X=Volume of blood to be pipetted 5x200/100=x X=10ms of blood Pipette 10mls of media, add 10mls of blood NB;Add blood at 45-50 ℃ (100 x 15 mm). List QC strains and expected growth

23. 5%BA Cont .. Sources of Error and Limitations Prepared medium should be used within the allowed/stated duration of weeks or months, or within the manufacturer’s reagent expiration date, whichever comes first. Expired medium should be discarded in a biohazard bin. Factors that adversely affect the expiration date and performance of prepared medium include: Overheating of base ingredients Using blood > 5 days old Adding blood or heat-labile ingredients at > 50°C Using improper storage temperatures Factors that adversely affect the expiration date and performance of prepared medium include: Storing medium in a frost-free refrigerator Under filling of medium container Sealing plates in storage bags before they have cooled and gelled properly Exposing medium to light Allowing the storage temperature of sealed medium bags to alternate from room temperature (22 to 25°C) to 2 to 8C Storing plates in open or improperly sealed bags (e.g., at setup or work benches) (unsealed bags result in a loss of medium volume that varies depending on the temperature and humidity of the room)

24. Preparation of CHO Prepare 200mls Use the formula RV/O=X R=Required concentration=5% V=Required volume=200mls O=Original conc of blood=100% X=Volume of blood to be pipetted 5x200/100=x X=10ms of blood Pipette 10mls of media, add 10mls of blood Add blood at 80-85 ℃ Organism ATCC Expected Results Haemophilus influenzae 10211 Growth Neisseria gonorrhoeae 43069 Growth

25. Preparation of MHA Prepare 200mls of media Read the manufactures instructions While using 15mmx90mm petri dish Pour between25-30mls media Growth of Quality control strains

26. Preparation of DNAse agar& CLED Prepare 200mls follow manufactures instructions Document results DNAse CLED

27. Preparation of MAC Prepare 200mls follow manufacture's instructions

28. ACTIVITY In your 9 groups 2 groups will prepare 1 media(BA,CHO,MAC,CLED&MHA) 9 th group will prepare DNAse Write a report and submit END ANY QNS????