SAN DIEGO MESA COLLEGE

Introduction to Microbiology Laboratory (Bio205) Instructor: Elmar Schmid, Ph.D.

I olation and tudy o! oil bact"ria

(En#iron$"ntal %n&no'n Lab) Laboratory Ob("cti#" After completion of this lab you should: 1. have gained a deep understanding of the important role of soil bacteria as decomposers and bio remediation agents in natural ecological cycles, e.g. carbon cycle, nitrogen cycle, on planet Earth !. have developed an appreciation for the important role of soil bacteria in decomposing "recycling# of the diverse forms of organic matter and biomass, including cellulose, hemicellulose, chitin, polycyclic aromatic hydrocarbons "PA$s#, phenolics, olefins, and other bio polymers %. have a good &no'ledge and gained s&ills in the applied methods and steps commonly used to isolate biomass degrading soil bacteria (. have a good 'or&ing &no'ledge 'ith different gro'th media and gro'th conditions ). be able to name and identify prominent biomass degrading soil bacteria Introduction *e often thin& of bacteria in a negative 'ay as disease causing +bad bugs,, but most people don-t reali.e that only a small fraction of bacteria are actually pathogenic. /he vast ma0ority of bacteria have chosen soil "and not the human body# as their preferred habitat 'here they play an enormously important ecological role as d"co$)o "r of biomass. /he top layers of the Earth-s terrestrial crust, often referred to as oil, is an enormously rich environment filled 'ith a plethora of different microbes, including bacteria most soils are comprised of inorganic geological materials, e.g. silica, minerals, degraded or decaying biomass and an e1tremely diverse biotic community 'hich comprises round'orms, proto.oans, fungi and soil bacteria 2any of these organisms, most importantly oil bact"ria, full fill important ecological functions. /hey play a pivotal role as decomposers of biological organic $at"rial (bio$a ) derived from either plants, animals or humans. Plant biomass includes cellulose and hemicellulose "found in leaves, stems, roots, etc.#, starch "found in fruits and tubers# and lignin "found as hardener in 'ood#. Animal biomass, e.g. derived from deceased animals or animal e1crements, include proteins and chitin "hair, bones, insect e1os&eletons#.

SAN DIEGO MESA COLLEGE

Introduction to Microbiology Laboratory (Bio205) Instructor: Elmar Schmid, Ph.D. Soil bacteria are not only able to brea& do'n components found in biomass but some are even capable to decompose components and chemicals found in certain human industrial or household 'astes, such as petroleum sludge, paper mill effluents and urea "in urine#. In the past decades, bacteria isolated from contaminated industrial soil and 'aste sites, became the center of increasingly important research in the field of bior"$"diation, i.e. the brea&do'n of industrial 'astes 'ith the help of life forms. 3or e1ample, certain bacteria, such as Enterobacteria sp., Bacillus subtilis, and Alcaligenes faecalis, 'hich 'ere isolated from soil e1posed to petroleum spilled from petrochemical plants, 'ere sho'n to be able to brea& do'n crude oil and even to1ic petroleum components, such as toluene, 1ylene and the recalcitrant polycyclic aromatic hydrocarbons "PA$s# naphthalene and anthracene. 2ost of the material found in rich soil is degraded biomass derived from green plants, mostly in form of cellulose. 4esides the glucose made polysaccharide c"llulo ", 'hich is the basic material of all plant derived biomass and forms the plants cell 'alls, degradation products of *"$ic"llulo ", tarc*, pectin, lignin and other phenolics are abundantly found in rich soils. Depending on the plant species, bet'een (5 6 758 of the plant material is cellulose 6 'hich after plant death or in form of dying leaves returns to the soil 'here it serves as important carbon source for the many decomposing microbes, including c"llulo "+d"grading (, c"llulolytic) bacteria. *hile a"robic oil bact"ria, e.g. Cytophaga, Archangium or Sporocytophaga, are found on the surface of soil particles usually close to the soil surface "'here they have easy access to o1ygen#, ana"robic oil bact"ria, most prominently Clostridia, prefer deeper soil depths 'here they can thrive in the absence of high o1ygen levels9 most soil bacteria are fre:uently found 'ithin smaller pores or crac&s of the soil particles, 'here they are less li&ely be eaten by predatory proto.oans on the 'ell aerated soil surface, the bacterial population density can reach bet'een 15; 6 15< cells per gram dry 'eight of soil= &eep this number in mind 'hen you are collecting your soil sample "you do not have to bring a buc&et of soil into the lab=# Scientists assume that based on comparative microbial D>A analysis data "P?@#, to date, only a minor portion "appro1imately 158# of the microscopically observable microorganisms in soil have been cultured and identified9

SAN DIEGO MESA COLLEGE

Introduction to Microbiology Laboratory (Bio205) Instructor: Elmar Schmid, Ph.D. /his section of the 4io!5) lab you 'ill be studying the presence of biomass degrading bacteria, 'ith a focus on c"llulo "+d"grading (, c"llulolytic) bact"ria, in soil samples you collected at different locations in San Diego county In preparation for this part of the course it is therefore important that you collect )5 6 155 g of biomass rich soil, compost material or of some form of decaying biomass "e.g. decomposing grass or hay# from a fairly 'ell researched collection site "e.g. study of surrounding plant andAor animal life, urban development, free'ay, runoffs, etc.# a couple of hours ahead of the scheduled lab9 this timely preparation allo's you to sieve "clean course metal sieve# the collected sample "'ithout contaminating it 'ith other bacteria, e.g. from your home environment of course9 &eep the sample sealed off# 'hen you collect the soil or biomass sample, decide ahead of time 'hether you 'ant to loo& for anaerobic soil bacteria "thriving in deeper soil sections# or 'hether you are interested to search for aerobic soil surface inhabiting bacteria protocol your collection site, date and sample characteristics "dry, moist, sandy, etc,#

Bou 'ill then: 1# enrich and isolate soil bacteria from sieved soil samples on special selection agar plates "C -rotocol A#, and in a parallel e1perimental set up !# observe bacterial cellulolytic activity in soil on cellulose filter discs plates "C -rotocol B# follo'ing the t'o protocols belo'.

-rotocol A. Enric*$"nt / i olation $"t*od

0"1uir"d Mat"rial / 0"ag"nt . ()"r tud"nt t"a$ o! t'o) )5 155 g of freshly collected soil, compost or decaying biomass samples "briefly air dried and sieved# small buc&et and sterile sieve or metal grid ( >utrient agar ">A# plates ( ?arbo1ymethyl cellulose "?2?# agar plates ! Starch nutrient agar "SA# plates ! S&im mil& nutrient agar "S2A# plates ! tubes 'ith Dlucose >utrient broth plus Durham tube "aerobic9 )ml# ! tubes 'ith Dlycerol >utrient broth plus Durham tube "aerobic9 )ml# 155 ml sterile 'ater "d$!E# in 1)5 ml glass bottle 'ith scre' cap 1 Sterile test tube "1)ml# 'ith scre' cap Sterile spatula or spoon Incubator set at %)o? "for the 'hole class# /able balance "plus sterile 'eighing boats# 1 Anaerobic cultivation pouch "44F# Sterile 15ml glass "or plastic# pipette "G pipette pump# Sterile 1ml glass "or plastic# pipette
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SAN DIEGO MESA COLLEGE

Introduction to Microbiology Laboratory (Bio205) Instructor: Elmar Schmid, Ph.D. -rinci)l". A freshly collected soil, compost or decaying biomass sample is suspended in sterile distilled 'ater by sha&ing and then is further diluted in distilled 'ater. An ali:uot of the undiluted and diluted sample is strea&ed onto the surface of four different agar plates. Ene set of the plates 'ill be incubated in an incubator under anaerobic conditions 'ith the help of an anaerobic pouch, 'hile the second set of agar plates 'ith the strea&ed bacterial samples is incubated under aerobic conditions. After % 6 7 days of incubation of the plates in an incubator at %)o?, students 'ill select and pic& individual colonies from the agar plates and start primary cultures for further analysis, characteri.ation and bacterial identification. -roc"dur". 1. *eigh in 15 g of your freshly collected soil, compost or other biomass sample ideal soil collection places are: forest soil, compost pile, la&e or river bed sediments, paper rich landfill areas H.any place 'ith lots of plant or paper litter collect the soil sample not longer than 1! hours before the start of the e1periments scoop the soil sample into a clean IipFoc bag and seal off !. Pour the soil sample into a bottle filled 'ith 155 ml sterile 'ater label this bottle 'ith your soil IDJ to avoid later mi1 up on the bench %. Suspend the soil sample into 'ater by sha&ing for about ) minutes (. Kse the sterile techni:ue and ma&e a 1:15 dilution of the suspended soil sample in 'ater9 to do this, pipette < ml of sterile 'ater into a clean, sterile 1) ml test tube "'ith scre' cap# and pipette 1ml of your suspended soil sample to this tube close cap and thoroughly mi1 the tube contents ). /riple strea& a loopfull of the undiluted soil sample onto the surface of t'o labeled >A plates "C )lat" 2 / )lat" 2# each label one plate "2# 'ith your soil ID J and also 'rite +undil.Aaerobic, on one plate label the second plate "2# 'ith your soil IDJ and 'rite +undil.Aanaerobic, L. /riple strea& a loopful of the 1:15 diluted soil sample onto the surface of a sterile >A plate "C )lat" 3# label plate "3# 'ith your soil ID J and also 'rite +dil.Aaerobic, on one plate 7. /riple strea& a loopful of the undiluted soil sample onto the surface of t'o labeled ?2? agar plates "C )lat" 4 / )lat" 5# label the plate ( 'ith your soil ID J and also 'rite +undil.Aaerobic, on this plate label the plate ) 'ith your soil IDJ and also 'rite +undil.Aanaerobic, on this plate ;. Sterile pipette 5.1ml of the undiluted soil sample into a Gluco " nutri"nt brot* tube and gently mi19 incubate this aerobic tube %) o? in an incubator for !( hours

SAN DIEGO MESA COLLEGE

Introduction to Microbiology Laboratory (Bio205) Instructor: Elmar Schmid, Ph.D. <. Sterile pipette 5.1ml of the undiluted soil sample into a Glyc"rol nutri"nt brot* tube and gently mi19 incubate this aerobic tube %) o? in an incubator for !( hours 15. Muic&ly place the plates 52 / 55 in bottom up fashion into the interior of an anaerobic pouch, add the chemical bag, seal off and then place the pouch into an incubator9 incubate for !( (; hours at %) o?9 11. Directly place the plates 526 3 / 4 in bottom up orientation in an incubator and incubate the plates for !( hours at %)o?9 1!. Denerally, incubate all the plates until clearly distinguishable bacterial colonies become visible on the agar surface "in most cases after 1 ! days# 1%. Pic& one colony from an aerobic plate and one colony from an anaerobic plate and triple strea& each colony on follo'ing plates for further isolation and characteri.ation >A plate Starch nutrient agar "SA# S&im mil& nutrient agar "S2A# plate ?2? agar plate label each plate accordingly 'ith your soil sample IDJ, name. colony, etc. 1(. Incubate the plates either aerobic "if pic&ed colonies 'ere from plates 1, % or (# or anaerobic "if colonies 'ere from plates ! G )# at %) o? for !( hours9 1). Ebserve the gro'n colonies on the plates and start doing further tests 'ith a selected colony for bacterial identification, e.g. gram staining, o1idase test, motility, etc. your instructor 'ill advise you 'ith 'hich biochemical tests you should continue

-rotocol B. Bact"rial c"llulo " d"gradation

0"1uir"d Mat"rial / 0"ag"nt . ()"r tud"nt) ?ollected soil samples "briefly sieved#, e.g. compost material and garden soil ! pieces of round *hatman filter paper >o. 1 " 7 cm# ! Sterile Petri dishes 4uc&et and sieve Sterile spatula or spoon Sterile forceps 3lat plastic container 'ith lid "filled 'ith 1 ! cm of distilled 'ater# Sprayer 'ater bottle Incubator -rinci)l".

SAN DIEGO MESA COLLEGE

Introduction to Microbiology Laboratory (Bio205) Instructor: Elmar Schmid, Ph.D. A crudely sieved soil sample is filled into a Petri dish, 'etted and covered 'ith a single piece of round filter paper "*hatman, >o.1#. /he thus prepared samples are incubated in a moist atmosphere "container# 'ithin an incubator over several days. Degradation of the cellulose components of the filter paper 'ill be indicated by the time dependent formation of color spots and degradation holes in the filter paper. ?ellulolytic microorganisms degrade the paper 'ith the help of cell secreted cellulase en.ymes. -roc"dur". 1. Sieve the collected soil sample through a crude sterile metal sieve to remove large debris !. 2oisten the sieved soil 'ith sterile distilled 'ater applied 'ith the help of a sprayer bottle %. Kse a sterile spoon or spatula and fill the 'etted soil into the ! sterile Petri dishes up to a height of 1 cm9 label the dishes accordingly, incl. soil sample ID J, date, etc. (. 3latten and smoothen the soil surface 'ith the help of another clean Petri dish ). Place a sterile *hatman filter paper >o.1 on the soil surface "use sterile forceps=# and thoroughly press the filter onto the soil L. Place the Petri dishes into an elevated section of the 'ater filled plastic container, put the lid onto the container and place it in an incubator ad0usted to %5 o? this design assured a moist atmosphere 'ithin the chamber throughout the incubation period ma&e sure to refill the chamber 'ith distilled 'ater from time to time 7. Incubate the Petri dishes for ) 6 7 days at %5 o? ;. ?hec& the *hatman filter paper in the Petri dishes for forming cellulose degradation 'hich 'ill be indicated by the formation of color spots "yello' orange red yello'Agreen# and of degradation holes Several microorganisms have been identified to be responsible for the observed cellulose degradation, including Cellulomonas "Eubacteria#, Cytophaga, Sporocytophaga, Sorangium "2y1obacteria# and a variety of cellulolytic fungi, e.g. /richoderma sp. "for a complete list of cellulolytic bacteria see 7abl" belo'#. Fab >otes:

SAN DIEGO MESA COLLEGE

Introduction to Microbiology Laboratory (Bio205) Instructor: Elmar Schmid, Ph.D.

7abl". Li t o! &no'n c"llulolytic oil bact"ria Bact"ria Na$" C*aract"ri tic >onsporulating, slender irregular rod shaped "coryneform# bacteria9 Dram )o iti#"9 A"robic or facultative anaerobic9 Eften motile9 ?onve1, yello' colonies
on peptone yeast agar

Ob "r#"d or I olat"d
"chec& mar&#

C"llulo$ona )8 e.g. C. cellulans e.g. C. fimi

?atalase positive9 ?ellulolytic bacteria

C. fimi produces beta 1,( endoglucanase "?enA gene#

Cyto)*aga )8 e.g. Polyangium

only cellulolytic species

/emp range: !) %)o? >on photosynthetic short cyclindrical rods9 Dram n"gati#" A"robic or facultative anaerobic9 2otility "gliding# *avy edged, depressed "agar# colonies9 sho' spherical or oval
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SAN DIEGO MESA COLLEGE

Introduction to Microbiology Laboratory (Bio205) Instructor: Elmar Schmid, Ph.D. S)orocyto)*aga )8 fruiting bodies '. slime envelope Bello', orange or red pigmented9 Decomposes proteins, starch, cellulase, pectin /emp. @ange: !5 6 %)o? >on photosynthetic gliding often sheathed bacterium9 Dram n"gati#"9 A"robic9 3orms yello', orange or red colored colonies9 Degradation of cellulose, carbo1ymethyl cellulose "?2?#, starch @od shaped, sporulating, motile bacterium9 Dram )o iti#"9 Obligat" ana"robic9 ?atalase negative9 often 'ith endospores ?ellulolytic bacteria
produce beta 1,( endo and e1oglucanases

9"r)"to i)*on

Clo tridiu$ )8 e.g. C. thermocellum "positive control bacterium in the 4io!5) lab# e.g. C. cellulolyticum e.g. C. cellobioparum

/emp. @ange: 15 L)o? Straight or curved rod shaped bacterium9 Dram n"gati#"9 A"robic "E! dependent#9 2otile9 ?atalase positive9 Produce fluorescent pigments "pyocyanins# ?ellulolytic9 /emp. @ange: %5o? ?ylindrical rod shaped bacteria9 Dram n"gati#"9 Strictly a"robic9 2otile "gliding#

- "udo$ona !luor" c"n : )8 c"llulo a

My;obact"ria )8 e.g. Sorangium cellulosum e.g. Myxococcus xanthus e.g. M. sp. ALe.g. Stigmatella aurantiaca

spread over agar surface "s'armers#

3orms orange or bro'n colored my1ospores and fruiting bodies 'ith sporangioles9 "optical refraction#9 Decompose cellulose "e.g. filter paper#9 ?ellulolytic and chitinolytic activity
produce beta 1,( endoglucanase "?elA gene# 8

SAN DIEGO MESA COLLEGE

Introduction to Microbiology Laboratory (Bio205) Instructor: Elmar Schmid, Ph.D. /emp. @ange: %5 %)o? Slender, rod shaped, non motile bacterium9 thermophilic9 Dram n"gati#"9 Obligat" a"rob"9 Does not gro' on nutrient broth, but on 5.18 casamino acids G 5.18 tryptone9 Digests cellulose G metaboli.es cellobiose, glucose, 1ylose, and galactose9 /emp. @ange: %7 75o? >onsporulating, motile, curved rod shaped bacterium9 Dram n"gati#"9 Obligat" ana"robic9 2otile9 3ermentative metabolism9 ?atalase negative9 Eften degrades cellulose, starch G other polysaccharides9 /emp. Eptimum: %5o? 2esophilic, rod shaped, usually non motile bacterium9 Dram n"gati#"9 Obligat" ana"robic9 Saccharolytic activity9 degrades crystalline forms of cellulose9 /emp. Eptimum: %)o? @od shaped, sporulating, motile bacteria9 Dram )o iti#"9 A"robic or facultatively anaerobic9 Ksually catalase positive9 Produce endocellulases
strains DFD, PAP11), and 1L;

Acidot*"r$u c"llulolyticu

Butyro#ibrio )8

Bact"roid" c"llulo ol#"n

Bacillu $"gat"riu$: ubtili

Str")to$yc"

)8

Sho' lichenoid leathery flat colonies and build hyphae as 'ell as air mycelia9 sporophores 'hich form N% spores9 3orm pigments "veget. cells O aerial mycelia#9 ?olonies 'ith typical soil smell9
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SAN DIEGO MESA COLLEGE

Introduction to Microbiology Laboratory (Bio205) Instructor: Elmar Schmid, Ph.D.
Deosmin production

e.g. Microbispora bispora e.g. Streptomyces !SM"# halstedii e.g. $hermospora fusca

Dram )o iti#"9 A"robic chemoheterotrophs9 ?atalase positive9 ?ellulolytic9

produces beta 1,( endoglucanase "?elA gene#

?ellulolytic9
produce beta 1,( endoglucanase "?asAA?elA1 gene#

?ellulolytic9
produces beta 1,( endoglucanase "E! gene#

0" ult 7abl". %n&no'n 2 ________________ Date Performed Test e%&% 'eth(l red _____________ Table # Result ne&at )e Comment a#ter 24 h ncubat "n n N* ____________________ Student Name Expected character !t c! "# dent # ed bacter um$

1+

SAN DIEGO MESA COLLEGE

Introduction to Microbiology Laboratory (Bio205) Instructor: Elmar Schmid, Ph.D.

$ acc"rd n& t" *er&e(,! 'anual "# Determ nat )e *acter "l"&( -9th Ed t "n.

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