CULTURE MEDIA & CULTURE METHODS

CULTURE MEDIA & CULTURE METHODS
STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala
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 – diffuse growth Solid medium – discrete colonies. STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Colony – macroscopically visible collection of millions of bacteria originating from a single bacterial cell. Cooked cut potato by Robert Koch – earliest solid medium Gelatin – not satisfactory - liquefy at 24oC STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Agar Frau Hesse Used for preparing solid medium Obtained from seaweeds. No nutritive value Not affected by the growth of the bacteria. Melts at 98oC & sets at 42oC 2% agar is employed in solid medium STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Types of culture media Based on their consistency a) solid medium b) liquid medium c) semi solid medium Based on the constituents/ ingredients a) simple medium b) complex medium c) synthetic or defined medium d) Special media STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Special media Enriched media Enrichment media Selective media Indicator media Differential media Sugar media Transport media Media for biochemical reactions Based on Oxygen requirement - Aerobic media - Anaerobic media STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Solid media – contains 2% agar Colony morphology, pigmentation, hemolysis can be appreciated. Eg: Nutrient agar, Blood agar Liquid media – no agar. For inoculum preparation, Blood culture, for the isolation of pathogens from a mixture. Eg: Nutrient broth Semi solid medium – 0.5% agar. Eg: Motility medium STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Simple media / basal media - Eg: NB, NA - NB consists of peptone, meat extract, NaCl, - NB + 2% agar = Nutrient agar STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Complex media Media other than basal media. They have added ingredients. Provide special nutrients Synthetic or defined media Media prepared from pure chemical substances and its exact composition is known Eg: peptone water – 1% peptone + 0.5% NaCl in water STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Enriched media Substances like blood, serum, egg are added to the basal medium. Used to grow bacteria that are exacting in their nutritional needs. Eg: Blood agar, Chocolate agar STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Differential media A media which has substances incorporated in it enabling it to distinguish between bacteria. Eg: Mac Conkey’s medium Peptone Lactose Agar Neutral red Taurocholate Distinguish between lactose fermenters & non lactose fermenters. STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Chocolate agar Chocolate agar Blood agar STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Enrichment media Liquid media used to isolate pathogens from a mixed culture. Media is incorporated with inhibitory substances to suppress the unwanted organism. Eg: Selenite F Broth – for the isolation of Salmonella, Shigella Alkaline Peptone Water – for Vibrio cholerae STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

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 STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

TCBS Mac Conkey’s medium TCBS STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Potassium Tellurite media LJ media STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Indicator media These media contain an indicator which changes its colour when a bacterium grows in them. Eg: Blood agar Mac Conkey’s medium Christensen’s urease medium STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

C.Diphtheriae on Potassium tellurite media STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Mac Conkey’s medium Urease medium STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Lactose fermenters – Pink colonies Non lactose fermenters – colourless colonies STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Sugar media Media containing any fermentable substance. Eg: glucose, arabinose, lactose, starch etc. Media consists of 1% of the sugar in peptone water. Contain a small tube (Durham’s tube) for the detection of gas by the bacteria. STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

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 STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Anaerobic media These media are used to grow anaerobic organisms. Eg: Robertson’s cooked meat medium, Thioglycolate medium. STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

TRIPLE SUGAR IRON AGAR (TSI) It is a composite media used to study different properties of a bacterium – sugar fermentation, gas production and H2S production. In addition to peptone, yeast extract & agar, it contains 3 sugars – Glucose, Lactose, Sucrose. The Iron salt – Ferric citrate indicates H2S production. Phenol red is the indicator. It is an orange red medium with a slant and a butt. pH of the medium – 7.4 STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Antibotics Probiotics Prebiotics Synbiotics STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Louis Pasteur ( ) Metchnikoff Elie ( ) Potential benefits of Lactobacillus~125 yrs ago 1905: Concept of Probiotics STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Colonization of Gut Starts immediately after birth Place of birth Type of Delivery Feeding: Time, Type Pre-lacteals vs Exclusive breast feed. Premature vs. Full term Sick babies STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Colonization of gut 1st Year;: > 200 bacterial species Adult : bacterial species Elderly: 300 Bact. Species Chr. Intestinal disorders STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Relative Proportion For context – Total Cells Theirs ~ 100,000 billion. Ours ~ 10,000 billion. STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Gut Flora in Breast Fed vs Formula Fed Babies

Friendly bacteria – Probiotics Unfriendly / harmful Bacteria – Pathogens STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Common flora Facultative Anaerobes Strict anaerobes Staph. Aureus, albus Nose & skin Mouth, Colon, Vagina Lactobacilus Sp, Bifidobact.. Candida albicans Mouth, Colon, Vagina Vagina, Outer urethra E. Coli. Haemophilus Sp. Nasophyrinx & Conjunctiva Pseudomonas aeruginosa Colon & skin STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala LACTOBACILLUS: BIFIDOBACTERIA: SACCHAROMYECES:

Probiotics ( “For Life’’) STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Probiotics (Friendly Bacteria) Normalise Intestine Immunomodulation
Metabolic effects Suppression of PPMs Strengthens immunity Production of vitamins; improves digestion Intestinal mucosal integrity Alleviate food allergy symptoms Lactose tolerance Regulation of bowel movement IBS Conrol of IBD Lowers cholesterol (Bile acid deconjugation &Secretion) STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Mechanism of Action Inhibit Potentially Pathogenic Microorganisms (PPMs) Reduction in Intestinal pH ( through production of SCFAs) Production of bacteriocins Competitive blocking of adhesion sites Competition for nutrients STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

MOA of Probiotics STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Types of Probiotics Most abundant Probiotic in GI Lactobacillus: Acidophilus, Rhamnosus, GG Plantarum, Reuteri, Bulgaricus, Sporogens Casei Action only in Small intestine STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Types of Probiotics Bifidobacteria 32 different species : Longum, Bifidum, infantis etc Most abundant probiotic next to lactobacilli Sp. in the GIT Action : Large Intestine STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Uniqueness of Bifidobacteria
Apart from the general Probiotics effect , Bifidobacteria helps is Glutamine synthesis Glutamine helps in maintaining the mucosal integrity NH3 + Glutamic acid > Glutamine Bifidobacteria STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Types of Probiotics Saccharomyces boulardii
Saccharomyeces: Boullardii, Salivarium, Thermophilus Non colonising yeast – so needs repeated readministration Action in large intestine STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Ideal properties of a Probiotic
1. Be nonpathogenic and nontoxic to the host 2. Be antagonistic to pathogens 3. Exert a beneficial effect on the host Capable of surviving, colonizing and proliferating in the gut (should not be killed by gastric juice / bile acids) 5. Able to inhabit in the S & L intestine 6. Must be of human origin 7. Contain a large number of viable cells and remain viable during storage and use STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Is Probiotic Prepn with Bifidobacteria Useful ?
Bifidobacteria is an Important Probiotic as it maintains the mucosal integrity Hence Bifidobacteria supplementation is useful in conditions like Gastroenteritis where the GI mucosa is severely damaged However, all the marketed preparation contains only 1 – 3 species of Bifidobacteria as against 32 required Hence it is ideal to supplement with probiotic which give nutrient to Bifidobacteria so all 32 species can proliferate STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Prebiotics Non-digestible dietary supplements, which provide ‘’nutrition’’ for Probiotics Oligosaccharides (fructo-oligosaccharides or FOS), Inulin, Lactulose, Lactitol Mutated Bacterial Species (Streptococcus faecalis, Clostridium butyricum, Bacillus mesentericus) Advantage of Prebiotics in bacterial form : Addl. Probiotic activity ( Intrinsic Probiotic activity) STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Prebiotics – Ideal Characters
Supply nutrient to beneficial bacteria Should escape digestion in the stomach and reach Intestine Should promote the proliferation of beneficial bacteria (Lactiobaccillus , Bifidobacteria) STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Prebiotics & Probiotics : Is there any difference ?
Nature of the Prep Microorganism Food supplement (eg: FOS) or Microorganism (eg : S.F ) Prime Fn To kill harmful pathogen To supply nutrition (Killing the pathogen is an additional effect) STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Prebiotics – Which is better ? FOS or live mutated bacteria ?
FOS – Recommended daily dose is gm Marketed prep. offer 100, 250 mg of FOS – Which is inadequate dose Also at high dose , FOS cause flatulence and GI discomfort Hence using a live mutated bacteria is beneficial as it would ensure the continuous colony count (nutrient) with out any side effect STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Pre/Probiotics - Indications 1. Infective diarrhea (viral, bacterial) 2. Antibiotic associated diarrhea 3. Lactose intolerance 4. Recurrent aphthous ulcers and stomatitis 5. Travelers’ diarrhea 6. Inflammatory IBD (Ulcerative colitis, Crohn’s) 7. Irritable bowel syndrome 8. Post operative state 9. Pouchitis 10. Diverticular disease of colon STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Infectious diarrhea Due to bacteria, Virus or Protozoa Viral diarrhoea : Rotavirus Mx : ORS / Infusion Bacterial Diarrhoea : E.coli , Salmonella , Shigella , V. Cholerae Mx : Antibacerial Protozoal Diarrhoea : E.Histolytics Mx : Metrinadozole An all the 3 types , there is a disturbance of the Intestinal microflora. Hence supplementation with Bifilac normalises the gut flora by displacing the PPMs and hence reduce the duration of diarrhoea STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Probiotics in prevention and treatment of diarrhea

Purported mechanisms of action
Free amino acids Organic acids Neutralization of dietary carcinogens β-Galactosidase activity Probiotics Oligosaccharides Immunostimulatory Cholesterol assimilation Antioxidant Short chain fatty acids Survival and adhesion competitions with pathogenic bacteria Bioactive peptides Bacteriocin STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Non-adhesive Adhesive M = M cells of intestinal epithelium L = Lymphocytes APC = Antigen presenting cells Th = T-helper cells IL = Interleukines TGF = Tumour growth factor IFN = Interferon TNF = Tumour necrosis factor Ig = Immunoglobulin Microorganisms Immune Response Intestinal Epithelium M L L L APC L Th0 IgG ↑ IgM ↑ IgE ↓ B Cell mediated response Antibody mediated response IL-2 ↑ IFN- γ ↑ TGF-β↓ IL-4 ↓ IL-10 ↓ + B Th2 Th1 IgA IL-2 ↑ IFN-γ ↑ TNF-α ↑ IFN-α ↑ Viruses Tumors Natural killer cells ↑ Macrophages ↑ Cytotoxic T-lymphocytes ↑ STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Potential mechanisms of probiotics for prevention or treatment of diarrhea Protection of intestinal epithelial barrier function Regulation of intestinal epithelial homeostasis Regulation of intestinal microbial environment Modifications to commensal and probiotic bacteria to enhance diarrhea prevention STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Antibiotic Associated Diarrhea Most common antibiotics that cause diarrhea Alteration in composition of normal intestinal bacterial micro flora by antibiotic makes the GI tract susceptible to infection by fungus (Candida) or bacteria, Clostridium difficile Fungus alters absorptive surface of GI tract – diarrhea STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala 24

Highest risk of AAD… Almost all antibiotics, particularly those that act on anaerobes, can cause diarrhea, but the risk is higher with aminopenicillins, a combination of aminopenicillins and clavulanate, cephalosporin's, and clindamycin. Diarrhea is a common adverse effect of antibiotic treatments. Antibiotic associated diarrhea occurs in about 5-30% of patients BMJ 2002;324: (8 JUNE) STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Probiotics decrease Diarrhea International Microbiology 2004 ; Mar 7(1) 59-62 STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Clostridium difficile Associated Diarrhea (CDAD)

Symbiotic Mixture of Pre and Probiotic Probiotics – Helps in reducing the PPMs Prebiotics – Helps in Providing food for Probiotics STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Bifilac Lactobacillus sporogenes 50 million ( Probiotic) Streptococcus faecalis T million ( Prebiotic) Clostridium butyricum TO-A 2 million ( Prebiotic) Bacillus mesentericus TO-A 1 million ( Prebiotic) STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Prebiotics in Bifilac Streptococcus faecalis T million ( Prebiotic) Clostridium butyricum TO-A 2 million ( Prebiotic) Bacillus mesentericus TO-A 1 million ( Prebiotic) STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Prebiotics – How do they get food ?
On ingestion, 3 mutated live bacteria continue to proliferate in the GI tract by a process of Symbiosis Symbiosis : Biological association of two or more species to their mutual benefit. Streptococcus faecalis ( Small ) Bacillus Mesentricus ( Small) Clostridium Butyricim ( Small & Large) STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

Thank you all STES, Sinhgad Institute of Pharmaceutical Sciences,Lonavala

CULTURE MEDIA & CULTURE METHODS

Similar presentations

Presentation on theme: "CULTURE MEDIA & CULTURE METHODS"— Presentation transcript:

Similar presentations

About project

Feedback

Log in

Auth with social network:

CULTURE MEDIA & CULTURE METHODS

Similar presentations

Presentation on theme: "CULTURE MEDIA & CULTURE METHODS"— Presentation transcript:

Similar presentations

About project

Feedback