QP code:780401
I Examination F.Y.B.Sc. Biotechnology Semester
Paper:BasicLifeSciences-||Microbia|Techniques
Model Answers
fifteen Q. 1. Do Y not 1. usuallY sPorefo gO% Time in minu 2. Decimal Reduction Time: e' or time in tinuitt tor the thermal dea population to doub|e in size 3. Generation time: The time taken for a 4'Exponentialpn,,"'Thephaseinwhichmicroorganismsgrowanddivideatmaxima| rate 5'Distortion:Anaberration/defectinmicroscopicimagewhichrendersasquare object as an image with curved sides' Acidic stain: Eosin/Acid fuschin/Nigrosine 6. acid/salts of Aluminium' iron' 7. Mordant: Gram's todine/ lodine solution/tannic coPPer,zinc,chromium 8. Detergent: 5"di,; lauryl sulfate/Catylpyridinium.chloride/soap 9.Anti-microbialA|dehyde:Formaldehyde/glutaraIdehyde 10. ChromoPhore 11. Chemotrophs factor 12. Vitamins/ growth plate/pour Hemocytometer count/spread 13. Microscopic methods- Breed's count/ plate/ Electronic coulter/ turbidostatic methods 14. NumericalaPerture 15. Nutrient agar/ 15. PhotograPhs 17. Incineration 18. lodoPhors 19. Death Phase 20. Abbe condenser (8 Marks) ., ,, Compound,, microscope and their app|ications Q2: a) Exp|ain : Simp|e,,and 4 marks each simple glass held usuallv in ff'$n:ilnstrument consisting of onty one lens or magnifving an adjustable frame oSimp|emicroscopesdonotgiveashighmagnificationasacompoundmicroscope. oApp|ication:tomakeanenlargedormagnifiedimageofminuteobjectsfor observations/ studY' Compound microscoPe: of lenses (1. Objective 2. EYePiece) . nn optical instrument consisting of Two sets and fine adjustment knobs, Stage, mounted in a holder/body tube' Coarse Condenser and Mirror . APPropriate diagram . Compound microscope gives much greater magnification of specimen due to product of magnification by objective (definite number of times) and further by the eyepiece . Application: for viewing minute objects such as bacteria/ tissue sections etc. by making a magnified image
Q 2: b) Discuss " Different types of Biological stains" with two examples from any three classes ( 7 amrks) Any three classes from o Nitro stains : Picric acid, Martius yellow, Aurantia o Azo stains: Methyl orange, methyl red, Brilliant yellow,Congo red, evan's blue, Sudan stains, trypan blue, vital red etc. o Anthraguinine stains: alizarin, alizarin red S., purpurin o Thiazole stains:Thioflavin S, geranine G, primuline, yellow G etc. o Quinonimine stains (lndamines, indophenols, thiamines, oxanines, azines): neutral red, neutral violet etc. o Phenyl methane stains (di/tri phenyl methane, diamino/triamino phenyl methane/ hydroxy phenyl methane, diphenyl-methyl methane): Brilliant green, Fast green, Methyl blue, methyl green Crystal violet, basic fuchsin, acid fuchsin etc. o Xanthene stains (pyronine, rhodamine, fluorane, phenolphthalein, sulfopthalein, acridine): PyronineB/Y, Rhodamine B, Eosine, Erythrosine, fluorescein, bromophenols, bromocresols, phenolphthalein, phenol red, acridine yellow/orange etc. o Natural stains: Indigo, Indigo carmine, Cochineal, Carmine, Orcein, Orcinol, LitmusBazilin, Hematoxylin etc.
Q 2: c) Describe the difference between " Light microscope" and "Dark field Microscope,, ( 8 marks) Minimum 4 correct four points
Light Microscope Dark field microscope
o Contrast between the object and the background is o The background of image in bright improved using a dark background /The specimens appears bright on a dark background o Abbe condenser o Dark-field stop in the condenser/ high aperture darkfield condenser (Abbe/paraboloid/cardiod) o Allows light to pass through the o Cone of light normally illuminating the object does object not enter the objective, only the light scattered or specimens reflected by the specimen seen by the objective o Used to view the objects visible in o Used to view the transparent/semitransparent a bright field (fixed/stained objects which are not readily visible in a bright field preparations) & State the functions of staining principle o{- "f r1m staining' Q 2: d) elaborate the th e orga ni sm s is ba sed o n a n d G ra m n e gative ;l :1ffi:'ltive lnm:,tmlu,:ru:i properties of the cell wall utt' differences in the physicochemicar !^Y.^ :::t::"' peptidog|ycan.,o,,tinrcngnetworkandlipidcontent.Cel|wallsofGramnegativeorgan|sms arethinner,have35%phospho|ipidsanaonty5-t5%peptidoglycanwithloosenetwork' Hence,crystalviolet-iodinecomplexformedau..ingthestainingisretainedbyGrampositive organismsandappearpurpleevenafterde-colourisationusingalcohol,whereasGram negativeorganismsaredeco|ourised.Counterstainingwitheosin/saffraninmakesGram pink' (3 marks) negative organisms appear
Staining solutions: ( 4 marks) cells violet/purple Basic stain: Stains all the (CVl) o CrystalViolet; forming crysta| vio|et. iodine Mordant ti"", tt,'" stain by o Gram,s iodine solution: comPlex oAlcohol:deco|ouriser:Removesthestainse|ectivelyfromGramnegativece|ls( Gram negative ) cells which counterstain, ,,r,n, ii" l".otou'i'eo o Eosin/saffranin: purple colour' positive organisms retain the appear pink 'Gram
Q.3a)Describeanyh^,omethodsbasedonUseofheatfordestructionofmicroorganisms ( 8 marks) ( 4 Marks each) Any two methods in detail: pressure: Autoclave o Moist heat/Stem under :Oven . Dry heat /hot air sterilization o lncineration inspissator e Tyndalization/fractional sterilization: o Pasteurization agent ( 7 marks) of an ideal ch-emical anti-microbial Q. 3b) Discuss properties any seven major properties from: Expected answer to explain 1' Antimicrobial activitY 2' SolubilitY 3. StabilitY other animals 4. Non-toxicity to human and 5. HomogeneitY organic material 6. Non-combination with extraneous at room or body temperature 7. Toxicity to microorganisms 8' CaPacitY to Penetrate 9. Non-corroding and non-staining 10. Deodorizing 11. Detergent caPacitY 12. AvailabilitY
Q.3c)Fi|trationcanbeusedse|ective|yforcontro|ofmicroorganismsforvarious examples (8 marks) appti"aiions ' lllustrate with earth pad in Seitz fi|ter, diatomaceous fi|ters of different materials -Asbestos Bacterio|ogical and sintered glass disks in other chamberland-pasteur filter in Berkefeld filter, porcelain in filters. Membrane or molecular filter - biologically inert cellulose esters Applications: Air sterilization : HEPA filter in laminar air flow proof filters sterilization of heat sensitive media and biological fluids - Bacteria Water filters etc. with q. 3d) Explain applications of heavy metals and dyes as anti-microbial agents examples (7 Marks)
( 4 marks) Heavy metals and their compounds have detrimental effects on microorganisms Examples: (anY 2 examPles) Mercurv: mercury- Used in Inorganic compounds : Mercuric chloride, Mercuric oxide, Ammoniated ointments disadv: toxicity and corrosive action less irritant and less organic compounds: mercurochrome, metaphen, merthiolate,mecresin: mucosal surfaces toxic; used as antiseptic preparations for cutaneous and
Silver : picrate- Used as antiseptic - colloidal silver compounds: silver nitrate, silver lactate, silver silver released from the combination Bacteriostatic/bacteriocidal effects depend on the free a gonococcal infection of eyes' Silver nitrate widely used to prevent opthalmia neonatorum,
Copper: .-_^r :_ . than bacteria-used in swimming^...i_. copper sulfate- More effective against algae and molds to prevent certain plant pools and open water reservoirs; Bordeaux mixture - afungicide diseases
Dyes anY two examples (3 marks)
the culture media on the basis of composition and Q.4A) Define culture medium. classify state any one use of each (8 marks)
preparation used to grow, transport or store Ans: The culture medium is a solid or liquid micro organisms. Classification of medium on the basis of composition: all the components are known is called 1) Synthetic or Defined medium: Medium in which Synthetic or Defined medium' It is use to Chemotrophs and heterotrophs' of unknown chemical 2) Complex medium: Medium which contains some ingredients medium may be sufficiently rich composition. such media are useful as a simple complex of many different micro organisms' and complete to meet the nutritional requirements To grow fastidious organisms' the organisms' S) Selective medium: Medium is used for selecting as it contains Na taurocholate' Mac conkey's agar selects the growth of enteric bacteria single organism from a group of organisms' 4) Differential medium: Medium differentiates and non hemolytic organisms' Blood agar is used to differentiate between hemolytic q'4 B) How would you cultivate microorganisms by chemostat and tubidostat (7 marks| chemostat: a chemostat contains sterile medium is fed into the culture vessel at the same rate as the media containing micro organisms is removed. The culture medium for the chemostat possesses an essential nutrients in the limiting quantities. Because of the presence of limiting nutrients, growth the rate is determined by the rate at which new medium is fed into the chamber and the final cell density depends on the concentration of the limiting nutrient. The rate of nutrient exchange is expressed as the dilution rate. lf teg dilution rate rises too high the organisms can actually be washed out of the culture vessel before reproducing because the dilution rate is greater. At very low dilution rate an increase in cell density and growth rate occurs. (4 marks) Turbidostat: lt has a photocell that measures the absorbance or turbidity of the culture in growth vessel. The flow rate of media through the vessel is automatically regulated to maintain pre a determined turbidity or cell density. lt provides a continuous supply of organisms in exponential phase. lt make possible to study the growth of organism,'und",. limiting nutrient supply. (3 marks|
q.4.c) write a detailed account on measurement of growth of microbes ( 7 marks) Ans: 1) Measurement of cell number: It can be done by Breed's count in which lsq. cm area sguare is marked on Grease free slide. Thin smear is made on slide. The smear is stained with crystal violet for 1min. Drain off the stain. Rinse the slide with d/w. observe and count organisms under oil immersion lens. Cell suspension is loaded on Hemocytometer slide. Either organisms in WBC chamber under low power or RBC chamber under high power are counted. 2) Measurement of cell mass: The cell mass can be measured by taking the packed cell volume of cells. The wet weight and dry weight of the cells is calculated. Spectrophotometer and colorimeter can be used to enumerate micro-organisms on the basis of turbidity of culture.
Q.4 D) How would you isolate pure culture by spread plate and streak plate techniques (7 Marks) Ans: Can be explained by drawing the techniques as well lsolation of pure culture by spread plate Method: 1. Suspension containing mixture of organisms is serially diluted. 2. Desired dilution amongst are selected. 3. 0.1 ml of the culture is added to sterile nutrient agar plate and spread on the surface of agar till the surface is dry, under sterile conditions. 4. Plates are incubated at desired temperature for desired time period depending on the nature and type of organisms. 5. After incubation time, well distributed and separated colonies appear. lsolation using streak plate: 1. The method involves- T- streak method: In this method, the sterile nutrient agar plate is divided into 3 zones- inoculation, sterile and isolation zone. A loopful of the culture sample is inoculated in inoculation zone and the culture is brought down perpendicularly in the isolation zone,where it is spread with the sterile nichrome Loop. Observe for the appearance of isolated colonies in the isolation zone after the end of incubation period. The other method is the Hexagonal method. In this method the medium plate is divided into 6 qudrants, taking care that the first and the sixth quadrants are not joint. lsolated colonies appear in the centre ofthe plate. Q.5 Short notes {any three) a) Halogens for microbial population control: lodine : Oldest and most effective germicidal agent Ti ncture iodine-several preparations lodophors-polyvinyl pyrrolidone (pvp-l) - non-staini ng and low irritant properties Applications: anti-bacterial, sporicidat, fungicidal, virucidal disinfectant; for skin; water; air; food utensils etc. Chlorine and chlorine compounds Chlorine gas: purification Hypochlorites: calcium hypochlorite, sodium hypochlorites Chloramines: monochloramine, chloramines-T, azochloramide Sanitizing agents, antiseptics
b) FDA method for evaluation of water soluble chemical disinfectant: Phenol-coefficient method is for determination of effectiveness of the test disinfectant (water soluble chemical disinfectant) as compared with that of pure phenol under the carefully standardized conditions. lt is based on cornparison of anti-microbial effects of the test disinfectant and phenol in liquid media inoculated with the same test microorganism under the same conditions. General procedure: o Series of dilutions of the test disinfectant o Similar series of dilutions of pure phenol of 1:90,1:90 and 1:100 o Temperature 2OoC o Inoculate 0.5 ml of standard young broth culture of the test microorganism o At regular time intervals, a loopful is transferred to 10 ml sterile broth o After 48 hours incubation at370c, growth in the broth tubes is recorded o lf all the organisms are killed : No growth in the corresponding subculture o Test is repeated with higher dilution of the disinfectant such that not all the organisms are killed in some of the tubes o Phenol coefficient is calculated as the ratio of the highest dilution of the test disinfectant not killing the organisms in five minutes (indicated by growth in the corresponding subculture) but killing in ten minutes (say it is 1:450) to the corresponding dilution of phenol (say it is 1:90, then Phenol coefficient for that disinfectant would be 450/90=5).
c) Zeil-Neelsen staining/ Acid fast staining: Differential staining used especially for staining Tuberculosis bacilli (Mycobacterium tuberculosis) and related organisms (genus: Mycobacterium) which have acid fast waxy material (mycolic acids) in the cell. The method is based on the relative solubility of the stains. Fuchsin is more soluble in phenol than in water or acid alcohol. phenol is more soluble in lipids/wax (that is present in the tubercule bacilli) than in water. Phenol with red fuchsin enters the cell lipids and remains even after treatment with decolourizing acid- alcohol.
Thin smear of cells /sample (i.e. sputum) is dried, heat-fixed and covered by carbol fuchsin solution and heated to gOoC over a steam bath for 4 minutes. This softens wax and the oye penetrates. Excess dye is washed off and smear is treated with alcohol containing s-i:O% hydrochloric acid to decolorize every other objects than the acid-fast bacteria. Methylene blue or Brilliant green is then applied as a counter-stain. Acid fast organisms appear bright red against the blue/green field. d) Arithmetic growth In certain abnormal condition s, the kinetics of bacterial growth may become arithmetic rather than exponential. A number of conditions can lead to arithmetic growth. For eg. lf a bacterium requires nicotinic acid as a growth factor is deprived of this nutrient, it is unable to synthesize pyridine nucleotide of which nicotinic acid is a specific biosynthetic precursor. As a result of the functions of pyridine nucleotide in electron transport chain, their levels in cell determined the overall rate of metabolism and hence growth. When no further synthesis of compound can occur, the growth rate of the population becomes directly proportional to the supply of pyridine nucleotide precursor in the cell. The rate of increase of cells remains constant. e) Phase -contrast microscope o Microscopy controls the contrast in the image in such a way that unstained cells/cytological details become visible ' Use of annular diaphram in the focal lane of the substage condenser, which controls illumination of the objects and phase shifting element/ phase plate o The light reflected by the object is passed through phase altering plate resulting in interference to reduce the illuminating rays in a systematic manner . Appropriate ray diagram o Used to view unstained cells/cytological details , advantage of accurate/ real-time viewing of unstained living cells /movements/propagation/exchange of cellular constituents and other processes etc.