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Culture Media


A.   Initially liquids (meat broth etc.) were used as culture media

  1. Disadvantages
    1. May not exhibit specific characteristics for identification
    2. Difficult to isolate different types of bacteria from mixed populations
  2. Advantages
    1. Used when large volumes used as inoculum (Blood, water)
    2. Preparing bulk cultures for antigens and vaccines
    3. Preparation of inoculum for biochemical reactions & antibiotic susceptibility testing

B.   Solid media introduced by Robert Koch

  1. Pieces of potato
  2. Gelatin (15%): liquefies at 24°C, broken by proteolytic bacteria
  3. Agar-agar
    1. Prepared from sea weeds
    2. Remains unmelted at all incubation temperatures
    3. Bacteriologically inert
    4. Does not add to nutritive properties of the medium
    5. Long chain polysaccharide, D-galactopyranose units
    6. Concentration used: 1-2%, M.P. : 95°C, S.P. : 42°C
  4. Peptone
    1. Water soluble products obtained from proteinaceous material e.g. lean meat, heart muscle casein, fibrin, soya flour
    2. By digestion with proteolytic enzymes pepsin, trypsin & papain
    3. Peptones, proteoses, amino acids, inorganic salts, accessory growth factors

(Recent Advances)

DPXDistrene 80  A synthetic resin) dissolved in Dibutyl phthalate (plasticizer) in Xylol is the mounting medium

Types of Culture Media

  1. Simple (basal), complex, synthetic or defined, special media: enriched, enrichment, 
  2. Selective, indicator, differential, sugar, transport media
  3. Solid, liquid, semisolid, liquid media
  4. Aerobic, anaerobic media 

A. Simple/ basal media

  1. Includes peptone water, nutrient broths and nutrient agar
  2. Nutrient broths: three types
    1. Meat infusion broth: aqueous extract of lean meat to which peptone is added
    2. Meat extract broth: mixture of meat extract (commercially prepared, aqueous extract of lean meat    concentrated by evaporation) with peptone
    3. Digest broth: aqueous extract of lean meat that has been digested with proteolytic enzyme so that additional peptone is not required
  1. Complex medium: have added ingredients for special purposes, bringing out certain characteristics, providing special nutrients
  2. Synthetic/ defined media: prepared from pure chemical substances & exact chemical composition of the medium is known; semisynthetic media: e.g. simple peptone water, 1% peptone with 0.5% NaCl
  3. Enriched media: Substances such as blood, serum, egg are added to the basal medium to grow fastidious bacteria. e.g. blood agar, chocolate agar, LSS.
  4. Enrichment media: when substance are added to liquid medium which inhibit the growth of the unwanted bacteria and favor the growth of the wanted bacteria. e.g. Tetrathionate broth, Selenite F broth, GN broth, alkaline peptone water.
  5. Selective media: when substance are added to solid medium which inhibit the growth of the unwanted bacteria and permit the growth of the wanted bacteria. e.g. MacConkey’s agar, DCA, Wilson Blair medium, XLD, bile esculin agar, mannitol salt agar.
  6. Indicator media: contain an indicator which changes colour when bacteria grow in them. e.g.  MacConkey’s agar (neutral red), DCA (neutral red), Christensen’s urea agar (phenol red), Simmon’s citrate medium (Bromo Thymol Blue), KTBA.
  7. Differential media: has substances incorporated in it, enabling to bring out differing characteristics of bacteria and thus helping to distinguish between them. e.g. MacConkey’s agar (LF and NLF), blood agar (alpha/beta/non-hemolytic),CLED (LF and NLF).
  8. Sugar media: fermentation of sugars is used for identification of various bacteria. Monosaccharide (arabinose, dextrose), disaccharide (lactose, sucrose), trisaccharide (raffinose), polysaccharide (starch), alcohols (glycerol), glucosides salicin,aesculin), no carbohydrates (inositol). Durham’s tube used to detect gas production    
  9. Transport media: used to maintain the viability of a pathogen and to avoid overgrowth of other contaminants during transit from the patient to the laboratory. e.g. Stuart’s, Amies transport medium, V.R. medium, Cary Blair medium.
  10. Storage media: used for maintenance of bacterial cultures. e.g. semisolid nutrient agar stabs, cooked meat broth, nutrient agar, blood agar, heated blood agar slopes, Dorset’s egg medium.  

B.  Solid / Liquid media

  1. Solod: Nutrient broth can be solidified by addition of 1-2% agar (Nutrient agar; solid medium)
  2. Semisolid agar: agar concentration 0.2-0.5%. used in testing motility of bacteria, Craigie’s tube for flagellar phase variation of Salmonella
  3. Hard/firm agar: agar concentration: 6%. Used for inhibiting swarming of bacteria
C.  Aerobic / anaerobic
Anaerobic media: used to grow anaerobic organism. Robertson’s cooked meat medium , thioglycollate broth.
Identification of Bacteria
  1. Catalase –
    1. on adding 3% H202 - Bubbles formation
    2. Used for identification of Gram Positive Cocci
    3. All Staphylococci - Positive
    4. Pneumo / Strepto / Entero - Negative
    5. Mostly aerobic bacilli - Positive
    6. Mostly Anaerobic Bacteria - Catalase Negative
  2. Oxidase
    1. On smearing a colony on reagent (Tetra methyl P-phenylene - diamine-dihydrochloride) --* purple color develops
    2. Oxidase positive - Neisseria, Moraxella, Pseudomonas, Aeromonas, Pleisomonas, Vibrio, Brucella
    3. Oxidase negative- Staphylococci, all entero bacteriacea members
  3. Indole-
    1. detects production of indole from tryptophan due to tryptophanase enzyme
    2. Indicator Kovac's reagent
    3. Positive in E.Coli , Negative in Klebsiella. Positive in Pr. Vulgaris and negative in Pr. mirabilis
    4. Positive Nitrosoindole - Cholera Red Reaction in V.cholerae  
  4. Citrate –
    1. detects the ability to utilize citrate as the sole source of carbon       
    2. Koser's medium (liquid) and Simmon's medium (solid),Bromothymol Blue (Indicator)
    3. If positive - medium turns blue from green and visible growth of bacterium in liquid/solid medium. Positive in Klebsiella and Pseudomonas, negative in E.Coli. 
  5. Nitrate –
    1. ability to convert nitrates to nitrites
    2. All members of family enterobacteriaceae are positive
  6. Urease –
    1. Christensen medium. This enzymes converts urea present in the medium to ammonia. Phenol red indicator turns red due to alkaline pH in Christensen’s urea agar. Indicator-bromothymol blue
    2. Positive in - H.pylori, Klebsiella, Proteus, Morganella, Staph, Brucella
  7. IMVIC reactions : Indole, Methyl Red, Voges Proskauer, Citrate
    E.Coli- ++--, Klebsiella- -- ++,  Salmonella- -+-+,  Shigella- -+--

Antibiotic Sensitivity

  1. Significance
    In the treatment and control of infectious diseases, especially when caused by pathogens that are often drug resistant, sensitivity testing is used to selected effective AMA.
  2. Limitations
    1.  It measures antimicrobial activity against bacteria under laboratory conditions (in vitro) not in patient (in vivo)
    2.  Selection also depends on patient’s clinical condition: any liver or renal disturbances, hypersensitivity, or any associated disease.
  3. Methods
    1. Dilution sensitivity test-broth or agar dilution
    2. Disc diffusion sensitivity test
    3. Modified Kirby-Bauer method and Stoke’s method
    4. Medium used: Muller Hinton agar
    5. Anaerobic: Wilkins Chalgren agar
    6. Lawn culture of test isolate
    7. Standard inoculums-McFarland turbidity standard-0.5
    8. Discs impregnated with a single high standardized concentration of the antibiotics
    9. Incubation temperature-37C. Exception- for detection of MRSA-33-35C
    10. Incubation time-16-18 hrs. Reading of results earlier/later may give wrong results.
    11. Control strains:
      a.  Staph aureus ATCC 25923, E.coli ATCC 25922, P aeruginosa ATCC 27853 
  4. B- Lactam (Penicillin) Resistance:
    B-lactams bind to penicillin binding proteins (PBP) which are present in cell membrane. PBPs or transpeptidase are enzymes responsible for cross bridge formation during cell wall synthesis. Hence their blockade will inhibit terminal step of peptidoglycan synthesis leading to Inhibition of cell wall synthesis.
B-Lactam resistance develops due to:
  1. Production of B-lactamases — open B-Lactam ring
  2. Alteration in penicillin binding proteins e.g. S. pneumoniae and MRSA (methicillin resistant Staphylococcus aureus)
  3. Genetically determined impermeability e.g. E.coli  
E. B - Lactamases: These are penicillin recognizing enzymes and structurally resemble PBPs. Thus instead of binding to PBPs drug binds to B-lactamases which then hydrolyze B-lactam ring.
  1. Site — B -lactamases are present in cell membrane in gram positive bacteria and also released outside bacterial cell.  Present in periplasmic space in gram negative bacteria and not secreted outside bacterial cell.
  2. Detection assays-
    a.  Acidimetric Method: Here drop in pH due to production of penicilloic acid from penicillin by B-lactamases is detected by phenol red indicator color change from red to yellow.
    b.  Iodometric method: in pH — intact penicillin molecule does not bind penicillin whereas penicilloic acid produced due to inactivation of beta lactam ring binds iodine. Reduction of Starch solution does not occur as iodine is unavailable in case of beta lactamase production and hence remains colorless.
    c.   Nitrocephin Test / Chromogenic cephalosporin test — Most sensitive — Smear a colony on nitrocephin disc change in color of disc from yellow to red means B - lactamase are present.
    d.  Standard dilution and disc diffusion assay  
  3. B-Lactamase Inhibitors Clavulanic Acid, Sulbactam, Tazobactam Mechanism of Action: resemble B-lactam antibiotics. Hence bind to beta lactamases reversibly or irreversibly thus protecting B-lactam antibiotic.  
  4. Extended spectrum B-lactamases ( ESBL) :
    These are enzymes which can hydrolyze even third generation cephalosporins and monobactam’s. Plasmid mediated enzymes. Plasmids responsible for ESBL production frequently carry genes encoding resistance to other drug classes (for example, aminoglycosides). Seen in Gram negative bacteria like E.Coli, Klebsiella, Pseudomonas etc. inhibited by B-lactamase inhibitors.
    An ESBL producer will be resistant to all oxyiminocephalosporines (Third generation cephalosporins like cefotaxime, ceftriaxone etc), Aztreonam and all penicillins. It is generally sensitive to cephamycins (like cefotaxime and cefotetan) and carbapenems (e.g. Imipenem and Meropenem)
  5. Detection by Double Disc Synergy test (DDS): Place a disc of amoxicillin + clavulanic acid on the side of disc containing cefotaxime or ceftazidime at a distance of 25 — 30mm on culture plate. T in zone of inhibition by —4 5mm or more suggests ESBL production.
  6. Vancomycin resistant Enterococci-
    1. NAMA and NAG form the major structural component of Gram-positive cell walls.
    2. Vancomycin forms hydrogen bond interactions with the terminal D-alanyl-D-alanine moieties of the NAM/NAG-peptides.
    3. This binding of vanco to the D-Ala-D-Ala prevents the incorporation of the NAM/NAG-peptide subunits into the peptidoglycan matrix.
    4. Mechanism of resistance to vancomycin appears to be alteration of the terminal amino acid residues of the NAM/NAG-peptide subunits to D-alanyl-D-lactate and D-alanyl-D-serine, as a result of which there is loss of affinity for the drug.
    5. 6 types Van A,B,C,D, E and F of which mainly A, B and C have been characterized.
    6. Van-A VRE is resistant to both vancomycin and teicoplanin
    7. Van-B VRE is resistant to vancomycin but sensitive to teicoplanin
    8. Van-C is only partly resistant to vancomycin, and sensitive to teicoplanin.
    9. Treatment for VRE- Linezolid

A patient having infection is empirically started on a cephalosporin and an aminoglycoside. Pus sent for culture reveals an ESBL producer. Course of action? AIIMS Nov 2010
A. Change the cephalosporin for a fluoroquinolone          
B. Change the cephalosporin for another cephalosporin
C. Change the cephalosporin for imipenem             
D. Don’t change the regime, just increase the dose


Ans - C

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