culture media

PowerPoint Presentation: 

In search of endodontic pathogens. . . . Microscopic examination Culture Immunological methods Molecular methods Kathmandu University Medical Journal (v-4,N-4,I-16, 2006)

PowerPoint Presentation: 

3

PowerPoint Presentation: 

CONTENTS INTRODUCTION TERMINOLOGY HISTORY ROOT CANAL FLORA PRINCIPLES OF CULTURING CULTURE MEDIA - REQUIMENTS - CONSTITUENTS - TYPES CULTURE METHODS - AEROBIC - ANAEROBIC

PowerPoint Presentation: 

MICROBIAL ROOT CANAL SAMPLING SAMPLING THE ABSCESS SAMPLING THROUGH SINUS METHODS OF ISOLATING PURE CULTURE REASONS FOR BACTERIAL UNCULTURABILITY CULTURE MEDIA FOR FUNGUS & VIRUSES LABORATORY TESTING FOR ANTIMICROBIAL SENSITIVITY ADVANTAGES & LIMITATIONS OF CULTURE METHODS CONCLUSION REFERENCES

PowerPoint Presentation: 

INTRODUCTION Accurate identification of micro organisms involved in a disease process is frequently essential not only for effective antimicrobial therapy but also for understanding the disease initiation & progression

PowerPoint Presentation: 

TERMINOLOGY CULTURING Process of propagating micro organisms in the laboratory by providing them with proper environmental conditions CULTURE MEDIUM combination of ingredients that will support the growth & multiplication of microorganisms by providing all the essential nutrients in order to cultivate these microorganisms in large numbers to study them.

PowerPoint Presentation: 

INOCULATION The process where a microbe of interest is introduced into a previously sterilized growth medium for the purpose of starting a culture of that microbe INCUBATION The provision of proper conditions for growth & development of bacterial or tissue culture.

PowerPoint Presentation: 

HISTORY 1 st BC :Varo & Columella – Diseases were caused by invisible beings 17 th Centuary (1683) – Antony Von Leeuwenhoek- oral microflora - root canal of severely decayed teeth little animal cules 1890: W.D. Miller - Father of Oral Microbiology – presence of bacteria with pulp disease .

PowerPoint Presentation: 

1909: EC Rosenow – Theory of focal infection- localized or generalized infection caused by bacteria traveling via bloodstream from distant focus of infection 1936: Fish & Mc Lean - pulps of healthy vital teeth were sterile 1939: Fish - Four distinct zones of periapical reaction in response to infection 1965: Kakehashi et al proved – bacteria – pulpal & periapical disease. 1976: Sundquist – Role of anaerobic bacteria in endodontic infection 1999: Baumgartner – predominant anaerobes in endodontic infection- P. nigrescens & P. intermedia

PowerPoint Presentation: 

MICROFLORA OF PULP SPACE Streptococci ὰ hemolytic streptococci- S.viridans-5% of total root canal flora 9% - non hemolytic streptococci 8% - S.salivarius Staphylocci - S.epidermidis Other Gm + ve bacteria – Pneumococci , Sarcinae , Lactobcilli , Bacillus subtilis , Diptheroids Gm – ve bacteria: Neisseria , Pseudomonas, E.coli , Veillonella & Bacteroids ( porphyromonas )

MICROFLORA OF PULP SPACE: 

MICROFLORA OF PULP SPACE Streptococci α hemolytic streptococci- S.viridans 5% of total root canal flora 9% - non hemolytic streptococci 8% - S.salivarius Staphylocci S.epidermidis Other Gm +ve bacteria pneumococci, Sarcinae, Lactobcilli, Bacillus subtilis, Diptheroids Gm –ve bacteria Neisseria, Pseudomonas, E.coli, Veillonella & Bacteroids (porphyromonas) Miscellaneous Candida Actinomyces Nocardia WJ Doughtery et al (1998, JOE)- most common in coronal & apical third- Flare-up Black pigmented bacteria-Porphyromonas melanogenicus, P.gingivalis, P.nigrescens &P.endodontalis Veillonella, Capnocytophaga, Eikenella, Bacteroids, Fusobacterium & Treponema. Traumatized but intact teeth with necrotic pulps B. melanogenicus Bacteria in periradicular space Facultative anaerobic streptococci Coliform rods Obligate anaerobes Acute infections of endodontic origin Bacteroides buccae, Bacteroids endodontalis, gingivalis & intermedius

PowerPoint Presentation: 

WJ Doughtery et al (1998, JOE)- most common in coronal & apical third- black pigmented bacteria- Porphyromonas melanogenicus , P.gingivalis , P.nigrescens & P.endodontalis Miscellaneous micro organisms: Candida Actinomyces Nocardia Traumatized but intact teeth with necrotic pulps – B. melanogenicus Acute infections of endodontic origin - Bacteroides buccae , Bacteroids endodontalis , gingivalis & intermedius

PowerPoint Presentation: 

Flare-up - Veillonella , Capnocytophaga , Eikenella , Bacteroids , Fusobacterium & Treponema . Bacteria in periradicular space Facultative anaerobic streptococci Coliform rods Obligate anaerobes Periapical abscesses Brook etal ( J.Perio 96) Fusobacteria nucleatum Prevotella intermedium Peptostreptococcus micros Porphyromonas gingivalis Streptococcus sanguis Streptococcus milleri

PowerPoint Presentation: 

Root filled teeth with apical periodontitis Molander et al 98, IEJ Enterococci Staph epidermidis Lactobacillus spp Fusobacterium spp E.Coli Association of specific bacteria with endodontic signs & symptoms Gomes & Drucker 94 (IEJ) With pain Gm- ve bacteria Gm + ve bacteria Prevotella spp Pepto streptococcus spp P.melanogenica

PowerPoint Presentation: 

Swelling: Gm- ve bacteria – prevotella spp , Fusobacterium necrophorum Gm+ve bacteria – Peptostreptococcus spp , P.micros Tenderness to percussion: Actinomyces meyeri Bacteroides gracilis L.Casei Prevotella denticola Purulent exudate : Fusobacterium spp , F. necrophorum , prevotells buccae , P.loeschii

PowerPoint Presentation: 

Bacterial species commonly found in asymptomatic infected root canals (53–55) ROBERT M. LOVE Endodontic Topics 2004, 9, 52–65 Gm + ve cocci Gm + ve rods Gm – ve cocci Gm – ve rods Streptococcus anginosus Actinomyces israeli Capnocytophaga ochracea Fusobacterium nucleatum S. sanguinis A. naeslundii C. sputigena Prevotella intermedia S.mitis P. melaninogenica S. mutans Eubacterium alactolyticum P. denticola E. lentum Veillonella parvula P. buccae Enterococcus faecalis E. nodatum P. buccalis E. timidum P. oralis Peptostreptococcus micros Campylobacter rectus P. anaerobius Propionibacterium propionicum C. curvus Porphyromonas gingivalis P. granulosum P. endodontalis Bacteroides gracilis Lactobacillus

PowerPoint Presentation: 

Bacteria Incidence % Fusobacterium nucleatum 48 Streptococcus sp 40 Bacteroides sp* 35 Prevotella intermedia 34 Peptostreptococcus micros 34 Eubacterium alactolyticum 34 Peptostreptococcus anaerobius 31 Lactobacillus sp 32 Eubacterium lentum 31 Fusobacterium sp 29 Campylobacter sp 25 Peptostreptococcus sp 15 Actinomyces sp 15 Bacteria Cultured and Identified from the Root Canals of Teeth with Apical Radiolucencies

PowerPoint Presentation: 

Bacteria Incidence % Eubacterium timidum 11 Capnocytophaga ochracea 11 Eubacterium brachy 9 Selenomonas sputigena 9 Veillonella parvula 9 Porphyromonas endodontalis 9 Prevotella buccae 9 Prevotella oralis Proprionibacterium propionicum 8 Prevotella denticola 6 Prevotella loescheii 6 Eubacterium nodatum 6

PowerPoint Presentation: 

PRINCIPLES OF CULTURE To determine the bacteriologic status of root canal system before obturation & assess the efficacy of debridement procedure To grow & isolate the microbial flora for antibiotic sensitivity & resistance profiles in cases of persistent infections 3. To determine the effectiveness of inter appointment medication & seal

PowerPoint Presentation: 

CULTURE MEDIA REQUIREMENTS Carbon-cellular constituents Nitrogen- proteins ATP Small amount of salts & trace elements- enzymatic activity CONSTITUENTS Water-hydrogen & oxygen Electrolyte- sodium chloride or other electrolytes

PowerPoint Presentation: 

Peptone – complex mixture of partially digested proteins. proteoses , aminoacids , polypeptides, phosphates, minerals, nicotinic acid & riboflavin Obtained from – lean meat, heart muscle, casein Meat extract – protein degradation products, inorganic salts, carbohydrates & growth factors Blood or serum : enrich culture media Agar: 2-3%. Act as solidifying agent

PowerPoint Presentation: 

TYPES OF CULTURE MEDIA ACCORDING TO FORM/CONSISTENCY OF MEDIA Solid media Liquid media Semisolid media ACCORDINT TO CONSTITUENTS OF MEDIA Simple media Complex media Synthetic or defined media Special media - Enriched media - Enrichment media - Selective media - Indicator or differential media - Sugar media - Transport media

PowerPoint Presentation: 

ACCORDING TO TYPE OF BACTERIAL GROWTH THEY SUPPORT Aerobic media Anaerobic media ACCORDING TO FUNCTION & USE Basal media – nutrient agar Complex media 1. Enrichment media 2. Selective media 3. Differential media 4. Transport media

PowerPoint Presentation: 

LIQUID MEDIA Wine or meat broth – Louis Pastuer Bacterial growth – change in broth’s appearance from clear to turbid Trypticase Soy Broth (TSB), Brain Heart Infusion Broth (BHI) Advantages Used for preparing bulk cultures for vaccines When large volumes are required for bacteria Disadvantages Bacteria may not exhibit specific characteristics for their diagnosis Difficult to isolate different types of bacteria from mixed populations

PowerPoint Presentation: 

SOLID MEDIA 1881- Robert Koch- potato, 2.5-5% gelatin & 1%meat extract. 1966- Moller : base culture medium- veal heart , peptone in agar gel SEMISOLID MEDIA Water Agar Growth enriching constituents: yeast extract & meat extract Blood: de fibrinated horse or sheep blood SIMPLE MEDIA Peptone water, 1% meat extract, sodium chloride & water Eg . Nutrient broth 2-3% agar + nutrient broth = nutrient agar

PowerPoint Presentation: 

COMPLEX MEDIA SYNTHETIC MEDIA prepared from pure chemical substances Used for special studies- metabollic requirements Eg . peptone water medium (1% peptone + 0.5% NaCl in water) ENRICHED MEDIA Blood, serum or egg + basal medium Eg . Blood agar, chocolate agar, egg agar

PowerPoint Presentation: 

ENRICHMENT MEDIA Tetrathionate broth- inhibits coliforms , allows growth typhoid bacilli DIFFERENTIAL MEDIA Substance incorporated in it – bring out differing characteristics of bacteria - helping to distinguish between them. Mc Conkey’s medium: peptone, lactose, agar, neutral red & taurocholate Lactose fermenters- pink colored colonies Non lactose fermenters- colour less or pale colonies

PowerPoint Presentation: 

TRANSPORT MEDIA Organisms that do not survive the time taken for transporting the specimens Eg . Stuart’s medium Buffered glycerol saline transport medium Reduced Transport Fluid Viability Maintaining Microbiostatic Medium III (VMGA III) Thioglycollate Medium

PowerPoint Presentation: 

SELECTIVE MEDIA Used to isolate particular bacteria from specimens - mixed bacterial flora Deoxycholate citrate agar- enteric bacilli Mac Conkey’s media – E.coli SUGAR MEDIA 1 % sugar in peptone water + andrade’s indicator-0.05%acid fuchsin in 1N NaOH ANAEROBIC MEDIA Robertson’s cooked meat media Thioglycollate broth

PowerPoint Presentation: 

AEROBIC CULTURE BROTH Ingredients of Brewer’s thioglycollate broth & their uses CONSTITUENT g/L Function Trypticase 15 Enrichment Cystine 0.5 Essential amino acid Dextrose 5 Carbohydrate Yeast extract 5 Vitamin & growth factor Sodium chloride 2.5 Keeps solution isotonic Sodium thioglycollate 0.5 Reducing agent Resazurin 0.001 Dye shows O2 presence with red band Agar 0.75 Prevents O2 diffusion

PowerPoint Presentation: 

Lag phase: Organisms adopt themselves to growth in medium , increase in size & metabolic activity. Stationary phase: Depletion of nutrients and accumulation of toxic products. When a bacterium is inoculated into medium, its growth follows a characteristic course.

PowerPoint Presentation: 

Culture characteristics of microorganisms of interest Streptococcus: Mitis salivarius agar - S.mutans: MSA+ Bacitracin high convex, opaque colonies - S.oralis : MSA Small, soft, non-adherent colonies - S.salivarius: MSA Large mucoid colonies - S.milleri : MSA+sulphonamide Small, non-adherent colonies 2. Lactobacillus casei , L.fermentum : Rogosa agar medium+ acetic acid

PowerPoint Presentation: 

3. Actinomyces : Blood agar medium A.odontolyticus : small round colonies with reddish brown center 4 . Micrococcus+staphylococcus : Blood agar medium Micrococcus mucilagenosus : large colonies adherent to surface of medium Staph.aureus : large, yellow pigmented colonies 5. Veillonella : Rogasa’s vancomycin agar 6. Haemophilus , parainfluenzae : Heated blood agar/ chocolate medium 7. Actinobacillus actinomycetemcomitans : Selective media containing vancomycin and bacitracin

PowerPoint Presentation: 

8. Eikenella corrodens : Blood agar medium corroding colonies on medium 9. B. gingivalis , Bacteroids intermedius : Blood agar medium Require vit K and haemin for growth. Black pigmented colonies Bacteroids oralis : Non pigmented colonies 10. Treponema denticola : Enriched media with serum T.macrodentium , T. ovale : colonization is poor 11. Entamoeba gingivalis : complex media 12. Candida albicans : Sabouraud’s medium- creamy white colonies 13. Mycoplasma orale ; M.salivarius : soft nutrient agar with 20 % serum, DNA, penicillin and thallus acetate.

PowerPoint Presentation: 

CULTURE METHODS Indications for culture: Isolate bacteria in pure culture Demonstrate their properties Obtain sufficient growth for preparation of antigens Determine sensitivity to antibiotics Estimate viable counts Maintain stock culture

PowerPoint Presentation: 

AEROBIC CULTURE METHODS Streak culture Lawn culture Stroke culture Stab culture Pour-plate culture Liquid culture STREAK CULTURE Isolation of bacteria in pure culture from clinical specimen.

PowerPoint Presentation: 

One loopful of specimen is smeared over the surface of agar plate The inoculum is distributed thinly over the plate by streaking it with the loop in series of parallel lines. On incubation, growth- confluent at the site of original inoculation, becomes progressively thinner & well separated colonies are obtained over the final series of streaks.

PowerPoint Presentation: 

LAWN / CARPET CULTURE Provides uniform surface growth of the bacterium Useful for antibiotic sensitivity test, antibacterial activity of materials Preparation of bacterial antigens & vaccines

PowerPoint Presentation: 

Prepared by flooding surface of plate with liquid culture- pipetting off excess inoculum- incubate the plate Applying swab soaked in bacterial culture STROKE CULTURE Made in tubes containing agar slope Employed for providing pure growth of bacteria for slide agglutination STAB CULTURE Puncturing nutrient gelatin or glucose agar with long, straight wire Demonstration of gelatin liquefaction & oxygen requirements of bacterium under study

PowerPoint Presentation: 

POUR PLATE CULTURE Tubes -15ml of agar medium-melted – cool in water bath 45-50C 1ml of inoculum + molten agar- poured into petri dishes- allowed to set Estimate the viable bacterial count in suspension

PowerPoint Presentation: 

LIQUID CULTURE Inoculated by touching with charged loop or by adding inoculum with pipettes or syringes Disadvantage: it does not provide pure culture from mixed inocula

PowerPoint Presentation: 

ANAEROBIC CULTURE METHODS Exclusion of oxygen / production of vaccum Displacement of oxygen with other gases 10% H2, 80% N2, He or 10% CO2 Absorption of oxygen by chemical (alkaline pyrogallol )or biological means ( germinating seeds) or reducing agents ( 0.1% thioglycolate , 0.1% ascorbic acid 0.05% cysteine , 1 % glucose)

PowerPoint Presentation: 

Most reliable & widely used anaerobic method- Mc Intosh-Flides anaerobic jar. Gaspak - disposable envelope-chemicals- generate H2 & CO2 on addition of water. H2 + O2 –anaerobic environment

PowerPoint Presentation: 

MICROBIAL ROOT CANAL SAMPLING ASEPTIC SAMPLE FROM ROOT CANAL Remove all hard & soft deposits from the tooth surface Isolate the tooth with rubber dam Apply 5 or10% iodine tincture or other antimicrobial agent all over the operation field Apply the iodine tincture neutralizer or other specific neutralizer Add sampling solution and execute pumping movements with a coarse file, pressing it against the root canal walls Take sample with charcolated paper points until all liquid is absorbed All points transferred to transport medium

PowerPoint Presentation: 

Inoculate into experimental media Incubation - anaerobic conditions at 37C Mandatory to apply neutralizers to prevent carry-over of the antimicrobial agents otherwise false negative results occur SUBSTANCE NEUTRALIZER CONCENTRATION Phenolic compounds Tween 80 1 % Mercury compounds Sodium Thioglycollate 0.05-0.1% Ammonium quaternary compounds Lecithin + Tween 80 0.5%+1.0% Chlorhexidine Lecithin + Tween 80 0.5%+1.0% Hypochlorites Sodium thiosulphate 1% Iodine Sodium thiosulphate 1%

PowerPoint Presentation: 

SAMPLING THE ABSCESS Fluctuant abscess is palpated, most dependent part of swelling determined Surface of mucosa – alcohol or iodophor Penetrated with 16-20 gauge needle, aspiration of exudates Aspirate injected into anaerobic transport medium SAMPLING THROUGH SINUS Orifice of sinus , surrounding mucous membrane dried with cotton rolls cleansed with 5-10% H2O2 Coarse paper point dipped in H2O2 – sinus for cleaning

PowerPoint Presentation: 

cleansed area - 5% iodine tincture-inactivated by 5% thiosulphate - 8-10sec Paper point introduced 10 sec. Transferred to culture medium, incubate- 10-14days Complex relationship present between MRS results & clinical outcomes Majority of cases (68%) healed with + ve culture at time of obturation, although frequency of healing was lower than teeth with negative culture (94%) Although Ca(OH)2 medication reduces no.of + ve cultures, this does not result in higher incidence of healing relative to teeth without this medication

PowerPoint Presentation: 

3. In culture studies of endodontically treated teeth with or without persistent lesions, a high proportion of healed cases still had + ve cultures, where as high proportion of teeth with persistent lesions had – ve culture. Why performance of MRS not particularly high? False negative Carryover effect of antibacterial irrigants or medicaments can inhibit bacterial growth during the cultivation process

PowerPoint Presentation: 

False positive MRS –technique sensitive procedure performed in a hostile setting Field of operation is not entirely sterile nor completely immune from saliva leakage & recontamination Bacterial status may change between appointments for incidental reasons unrelated to study itself. Incidental false negative Bacteria die off- nutrient deprived root canal environment MRS measured bacterial status correctly-results will be incorrect- impossible to determine whether it is actually incorrect

PowerPoint Presentation: 

Incidental false positive Bacteria can re enter root canal system between visits through coronal leakage of the temporary restoration and/ or marginal deficiency, cracks & exposed dentinal tubules Anachoresis How useful is root canal culturing in predicting treatment outcome? Chankhrit Santhorn et al JOE- Vol-33, N-3, Mar 2007

PowerPoint Presentation: 

What Treatment Does What Bacteria Have to Do to Survive Mechanical effect: removal by instruments Colonize areas distant from the main canal ( eg , isthmus, ramifications, and dentinal tubules) Chemical effect: irrigation Colonize areas distant from the main canal ( eg , isthmus, ramifications, and dentinal tubules); Be protected by tissue remnants, dentin, serum or dead cells, all of which have the ability to inactivate or reduce the efficacy of antimicrobial agents; Be intrinsically resistant to the antimicrobial agent Form biofilm structures enclosed by a protective polysaccharide matrix Chemical effect: inter appointment medication Be protected by tissue remnants, dentin, serum or dead cells, all of which have the ability to inactivate or reduce the efficacy of antimicrobial agents; Be intrinsically resistant to the antimicrobial agent Form biofilm structures enclosed by a protective polysaccharide matrix Clinician versus Bacteria: The Bacteria Way of Deceiving Treatment

PowerPoint Presentation: 

Ecological effect: nutrient deprivation Adapt to the new environment, turning on survival genes and alternative metabolic pathways; Enter a viable but noncultivable state Be located in areas where nutrient sources were relatively unaffected (very apical part of the canal near the foramen, ramifications) Studies that Identified Bacteria Persisting after Intracanal Disinfection Procedures study irrigant Sample taken after Identification method Frequent taxa Gm+ve bacteria Byström & Sundqvist Saline Chemomechanical preparation Culture Peptostreptococcus anaerobius Parvimonas micra Lactobacillus spp. Bacteroides spp. 70% Byström & Sundqvist 5% NaOCl Chemomechanical preparation Culture Streptococcus intermedius Fusobacterium nucleatum 50%

PowerPoint Presentation: 

study irrigant Sample taken after Identification method Frequent taxa Gm+ve bacteria Byström & Sundqvist 0.5% NaOCl Chemomechanical preparation Culture Fusobacterium spp. Streptococcus spp. Eubacterium brachy Lactobacillus spp. Porphyromonas gingivalis Prevotellaintermedia 45% Byström & Sundqvist 5% NaOCl EDTA Chemomechanical preparation Culture Streptococcus spp. 75% Sjogren & Sundqvist 0.5% NaOCl Chemomechanical preparation Culture Fusobacterium nucleatum Parvimonas micra 67%

PowerPoint Presentation: 

study irrigant Sample taken after Identification method Frequent taxa Gm+ve bacteria Sjogren etal . 0.5% NaOCl Chemomechanical preparation+10min Ca(OH)2 Culture Fusobacterium nucleatum 43% Gomes et al. 2.5% NaOCl EDTA Chemomechanical preparation Culture Streptococcus anginosus group Parvimonas micra Lactobacillus acidophilus 80% Sjogren etal 0.5% NaOCl Chemomechanical preparation Culture Pseudoramibacter alactolyticus Fusobacterium nucleatum Campylobacter rectus Parvimonas micra 62%

PowerPoint Presentation: 

study irrigant Sample taken after Identification method Frequent taxa Gm+ve bacteria Peters et al. 2% NaOCl Chemomechanical preparation+10min Ca(OH)2 Culture Actinomyces odontolyticus Prevotella intermedia Parvimonas micra Eggerthella lenta Prevotella oralis 58% Peters et al. 2% NaOCl Intracanal medication– Ca(OH)2 Culture Propionibacterium acnes Parvimonas micra Veillonella spp. Bifidobacterium spp. Capnocytophaga spp. 61% Chavez de Paz et al 0.5% NaOCl Intracanal medication– Ca(OH)2 Culture Lactobacillus spp. Streptococcus spp. Enterococcus spp. Propionibacterium spp. 88%

PowerPoint Presentation: 

study irrigant Sample taken after Identification method Frequent taxa Gm+ve bacteria Kvist et al. 0.5% NaOCl Chemomechanical preparation Culture Streptococcus spp. Peptostreptoccus spp. Prevotella spp. 71% Kvist et al. 0.5% NaOCl Intracanal medication– Ca(OH)2 Culture Staphylococcus spp. Streptococcus spp. 90% Chu et al. 0.5% NaOCl Intracanal medication– Ca(OH)2 Culture Neisseria spp. Staphylococcus spp. Capnocytophaga spp. Actinomyces spp. 60% Vianna et al 2% CHX (gel) Chemomechanical preparation Culture Propionibacterium acnes Propionibacterium propionicum 82% Vianna et al 2% CHX (gel) Intracanal medication– Ca(OH)2 Culture Propionibacterium acnes 80%

PowerPoint Presentation: 

study irrigant Sample taken after Identification method Frequent taxa Gm+ve bacteria Vianna et al 2% CHX (gel) Intracanal medication– CHX GEL Culture Gemmella morbillorum Clostridium argentinense 91% Vianna et al 2% CHX (gel) Intracanal medication– Ca(OH)2/CHX Culture Gemmella morbillorum 78% Review Article Clinical Implications and Microbiology of Bacterial Persistence after Treatment Procedures José F. Siqueira etal JOE — Volume 34, Number 11, November 2008

PowerPoint Presentation: 

METHODS OF ISOLATING PURE CULTURE Surface plating Pre treatment of specimens with appropriate bactericidal substances which destroy unwanted bacteria Obligate aerobes & anaerobes may be separated by cultivation under aerobic or anaerobic conditions Separation of bacteria with different temp optima can be effected by incubation at different temp. Bacteria of differing sizes may be separated by the use of selective filters

PowerPoint Presentation: 

REASONS FOR BACTERIAL UNCULTURABILITY Lack of essential nutrients or growth factors in the artificial culture medium Over feeding conditions Toxicity of the culture medium itself, which can inhibit bacterial growth Production of substances inhibitory to the target micro organisms by other species present in a mixed consortium Metabolic dependence on other species for growth Bacterial dormancy: bacteria unable to divide or form colonies on agar plates

PowerPoint Presentation: 

Culture media for fungi Potato dextrose agar Cornmeal agar Malt agar Sabouraud’s agar Antifungal efficacy of 5.25% NaOCl, 2% CHX & 17% EDTA with & without an antifungal agent Saurabh S. chandra etal JOE V-36, N-4, April 2010 65 human mand pm – decoronated at CEJ – WL determined – apical preparation done #50 K file – 5.25% NaOCl, 17% EDTA

PowerPoint Presentation: 

Root – nail varnish. Apical foramen sealed – type II GIC –autoclave C.albicans (1.5* 10 8 / mL ) – 0.5ml-teeth-stored glass test tube – 37C, 91% humidity -96hrs After 48hrs – sample taken from tooth with syringe, plated on Sabouraud dextrose agar – to verify the growth of C.albicans After 96 hrs – 30 teeth – 2 ml irrigants – 1min - 5 ml distilled water. 30 teeth- irrigants + 2ml 1 % clotrimazole -1 min – distilled water Hand filing done – 1um inoculation loop – remove sample from canal- SDA -37C- 48hrs NaOCl> 2%CHX & 17 % EDTA NaOCl & 2% CHX with clotrimazole > 17 % EDTA with clotrimazole

PowerPoint Presentation: 

Cell culture for viruses Cell culture involves growing cells as monolayer sheets upon a glass surface. Primary cell culture Diploid cell strains Continuous cell strains Primary cell cultures : These are normal cells freshly taken from the body and cultured. They are capable of limited growth and cannot be maintained in serial culture. E.g. - Monkey kidney, human embryonic kidney, human amnion and chick embryo cell cultures

PowerPoint Presentation: 

Diploid cell strains : These are cells of a single type that retain the diploid chromosome number during serial of cultivation for a limited number of times. E.g. - human embryonic lung cell strain, rhesus embryo cell strain. Continuous cell strains : These are cells of a single type, usually derived from cancer cells that are capable of continuous serial cultivation indefinitely. These cell lines are stored in the cold (-70 O C) for use when necessary. E.g. - Baby hamster cell line, L 929 mouse fibroblast cell line etc

PowerPoint Presentation: 

LABORATORY TESTING FOR ANTIMICROBIAL SENSITIVITY Kirby Bauer disc diffusion method Disc diffusion method Stokes disc diffusion method Petri dish size- 100mm in diameter. Medium – depth of 4mm (25ml) pH of medium: 7.2 & 7.4 Density of organisms- 1.5* 10 8 cfu /ml = 0.5 Mc Farland standard Mc Farland 0.5 – 9.95ml of 1% sulphuric acid & 0.05ml of 1% barium chloride Antibiotic disc – 6mm in diameter

PowerPoint Presentation: 

KIRBY-BAUER DISC DIFFUSION METHOD Muller Hinton Agar plate – swabed with inoculum - 3-5min Antimicrobial impregnated discs placed on the surface of agar- placed not closer than 24mm Seven discs- one at centre – 6 in the periphery – 35-37C- 16-18hrs Zone of inhibition- caliper/ transparent plastic ruler

PowerPoint Presentation: 

STOKES DISC DIFFUSION METHOD Plate -3 parts- test organism central one third- control on upper & lower thirds 6 antibiotic discs – 35-27C -16-18hrs

PowerPoint Presentation: 

Zone size interpreted as: Sensitive : zone size equal to, larger than/ not more than 3mm smaller than control Intermediate : size- 2mm Resistant : size < 2mm MINIMUM INHIBITORY CONCENTRATION MIC – Least amount of antimicrobial agent that inhibits visible growth of an organism after over night incubation MBC – Minimum concentration of drug that kills 99.9% of the test organism in the original inoculum

PowerPoint Presentation: 

Antimicrobial agent incorporated into culture medium- 0.25, 0.5,1,2,4,8,16,32,64,128 mg/ml. Broth -inoculated with suspension of test organism –37C-16-18hrs Standard inoculum from test tube-no growth occurred- subcultured on blood agar to determine the minimum conc of drug required to kill the organism (MBC)

PowerPoint Presentation: 

DIFFERENT STRAINS OF BACTERIA USED IN CULTURE STUDIES F. nucleatum (ATCC 10953) (ATCC 29212) S.sanguinis (ATCC 33397) (ATCC 10556) Candida albicans (ATCC 10556) C627 Staphylococcus aureus (ATCC 25923), Prevotella intermedia (ATCC 33277) Actinomyces naeslundii (ATCC 49340) Enterococcus faecalis (ATCC 49046) (ATCC 29212) (ATCC 19434) (ATCC 4082) VP3- 181, GEL 31, and GEL 32

PowerPoint Presentation: 

Staphylococcus epidermidis C621 S.mutans – ATCC 10449, ATCC 25175 S.sobrinus –NCTC 6715, ATCC 33478 L.acidophilus – ATCC 4356 A. viscosus – ATCC 43146 S.cricetus – ATCC 19642 Porphyromonas gingivalis (ATCC 10953) Escherichia coli C498

PowerPoint Presentation: 

Antibacterial effect of MDPB against anaerobes associated with endodontic infections IZUTANI etal Gm+ve coccus - E.faecalis SS 497- BHI agar Gm- ve rods: Fusobacterium nucleatum 1436- Todd Hewitt broth+ 0.05% L cysteine hydrochloride monohydrate Prevotella nigrescens ATCC 33563- BHI broth+ 0.05% L cysteine hydrochloride monohydrate+ 1% haemin , 0.02% vit K3 Agar disc diffusion test: Bacteria cultivated anaerobically – 12hrs 350ul bacteria suspension spread on agar plate

PowerPoint Presentation: 

20ul of MDPB impregnated filter paper discs( 6mm, 1.5mm thick) placed on agar plate Incubation – 37C – 48hrs – E.faecalis, F.nucleatum 96hrs – P. nigrescens Size of inhibition zone = (I-F)/2 I = mean of three measurements of diameter of inhibition halo F = diameter if filter paper (6mm) MIC/MBC MEASUREMENT Conc of MDPB- 0.24 – 500 ug / mL

PowerPoint Presentation: 

Bacterial suspension – 2*10 6 CFU/ml – 50uL of it inoculated into well containing MDPB solution – 37C- 48hrs-E.faecalis, F.nucleatum, 96 hrs – P. nigrescence MIC values – least conc in the well at which turbidity was not observed by visual examination IEJ 43, 2010, 637-45

PowerPoint Presentation: 

Evaluation of antimicrobial efficacy of 0.5% IKI, 3% NaOCl &0.2% CHX when used alone & in combination as intracanal irrigants against E.faecalis: An invitro study Neera joshi etal 64 single rooted teeth- sterilization autoclave E.Faecalis inoculated – 24hrs 8 groups – irrigation – 30 sec Sterile round bur-1mm diameter- hole was drilled from proximal surface of tooth into root canal Dentinal shavings allowed to fall on BHI agar plate Incubation – 48hrs – 36.5C IKI- best antimicrobial effect – middle third of root canal CHX+ NaOCl – apical third ENDODONTOLGY,V-21,I-2,DEC 2009

PowerPoint Presentation: 

In Vitro Evaluation of the Antimicrobial Effects of a RootCanal Sealer-Antibiotic Combination Against Enterococcus faecalis Anita A. Hoelscher et al JOE — Volume 32, Number 2, February 2006 Purpose : To evaluate the antimicrobial effects of five antibiotics when individually added to Kerr EWT sealer against E. faecalis. Sterile paper discs, 6 mm in diameter, were saturated with one of the sealer-antibiotic combinations by exposing the paper discs to the sealer mixture 15 plates were incubated aerobically and the remaining 15 plates were incubated anaerobically using an Anaerobic GasPak jar All 30 BHI plates were subject to incubation at 37°C for 48 h and the diameter of the zones of inhibition were measured in mm

Mean zones of inhibition (millimeters) for sealer-antibiotic combinations and Kerr EWT sealer, for aerobic and anaerobic groups combined: 

Mean zones of inhibition (millimeters) for sealer-antibiotic combinations and Kerr EWT sealer, for aerobic and anaerobic groups combined Amoxicillin - largest zone of inhibition- 31.6 mm. penicillin (26.9 mm), doxycycline (19.6 mm) clindamycin (13.9 mm), and metronidazole (0.6 mm). Kerr EWT sealer control group was 0.7 mm. No zone of inhibition - blank sterile paper disc controls.

PowerPoint Presentation: 

Comparison of the antibacterial efficiency of neem leaf extract & 2% NaOCl against E.faecalis, C.albican & mixed culture – an invitro study Aarti bohora et al ENDODONTOGY V-22,I-1 JUNE2010 E.faecalis, C.albicans – BHI broth – 37C-24hrs 200uL culture spread on BHI agar plate Mixed culture – 1:1 – 200uL Wells of 6mm diameter made in agar surface 50 uL neam leaf extract, NaOCl & control- added to wells – 37C-24hrs Zone of inhibition recorded in mm

PowerPoint Presentation: 

ADVANTAGES & LIMITATIONS OF CULTURE METHODS ADVANTAGES Broad range nature, identification of unexpected species Allow quantification of all major viable micro organisms in the samples Allow determination of antimicrobial susceptibilities of the isolates Pathogenecity studies are possible Widely available

PowerPoint Presentation: 

LIMITATIONS Impossibility of culturing a large no.of extant bacterial species Not all viable bacteria can be recovered Once isolated, bacteria require identification using no.of techniques Low sensitivity Strict dependence on the mode of sample transport Samples require immediate processing Costly, time consuming & laborious Specificity dependent on the experience of the microbiologist Extensive expertise & specialized equipment needed to isolate strict anaerobes Take several days to weeks to identify most anaerobic bacteria

PowerPoint Presentation: 

CONCLUSION Main purpose of culturing clinically is to serve as predictor of healing The empirical administration of antibiotics may not produce satisfactory results, in such cases culturing may provide a valuable information for better antibiotic selection

PowerPoint Presentation: 

Endodontics- Ingle & Bakland 5 th edition Pathways of Pulp - stephen cohen 8 th edition Endodontic practice- Grossman Text of microbiology- Anantanarayan R - 6 th ed. Text book of microbiology – Arora Endodontics prep manual for under gratuates : Jayshree Hegde Endodontic Science- Carlos Estrela-vol-1,2 ed Invasion of dentinal tubules by root canal bacteria ROBERT M. LOVE Endodontic Topics 2004, 9, 52–65 In search of endodontic pathogens Suchitra , Kundabala , Shenoy Kathmandu University Medical Journal (2006), Vol. 4, No. 4, Issue 16, 525-529 How Useful Is Root Canal Culturing in Predicting Treatment Outcome? Chankhrit Sathorn etal JOE—Volume 33, Number 3, March 2007 REFERENCES

PowerPoint Presentation: 

84 84 T H A N K Y OU