Matriculation (Malaysia) complete lab report of Physics Experiment 5.
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inorganic chemistry
How GC is coupled to MS to determine trace analysis compounds in waterFull description
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EXPERIMENT 5 Diferential Staining Technique echniqu e Simple staining depends on the act that acterial cells difer chemicall! rom their surroundings and thus can e stained to contrasts "ith their surroundings# Microorganisms also difer rom one another chemicall! ph!sicall!$ and thus ma! react diferentl! to a gi%en staining procedure# This is the asic principal upon "hich diferential staining depends# Thus$ "e ma! &nd diferential staining as a method o distinguished et"een t!pes o acteria#
A. The Gram Stain The 'ram Stain$ the most useul staining procedure in acteriolog!$ is a diferential stain# In this procedure acteria are di%ided into t"o groups# The &rst o these groups is stained purple ! the gram stain$ "hile the second group is stained pin(# The organisms stained purple "as called gram) positi%e* the organisms stained pin( is the gram)negati%e# +r!stal %iolet is &rst applied$ ollo"ed ! the mordant iodine$ "hich &,es the stain# Then the slide is "ashed "ith alcohol$ and the 'ram-positi%e acteria retain the cr!stal-%iolet iodine stain* ho"e%er$ the 'ram-negati%e acteria lose the stain# The 'ram-negati%e acteria susequentl! stain "ith the saranin d!e$ the counterstain$ used ne,t# These acteria appear red under the oil-immersion lens$ "hile 'ram-positi%e acteria appear lue or purple$ re.ecting the cr!stal %iolet retained during the "ashing step#
/nother diferential stain technique is the acid-ast technique# This technique diferentiates species o Mycobacterium rom Mycobacterium rom other acteria# 0eat or a lipid sol%ent is used to carr! the &rst stain$ carol uchsin$ into the cells#
Then the cells are "ashed "ith a dilute acid-alcohol solution# Mycobacterium species resist the efect o the acidalcohol and retain the carol uchsin stain 1right red2# 3ther acteria lose the stain and ta(e on the susequent meth!lene lue stain 1lue2# Thus$ the acid-ast acteria appear right red$ "hile the nonacid-ast acteria appear lue "hen oser%ed under oil-immersion microscop!#
OBJECTIVE
To learn the gram staining method and to oser%e the characteristic o gram)positi%e acteria and gram)negati%e acteria#
4# / separate thin smears o E.Coli, Staphylococcus aureus and Bacillus subtilis "as prepared# It "as air dried and heat &,ed# # The smears "ere co%ered "ith cr!stal %iolet or one minute# 9# In a slo" running tap "ater$ the smears "ere rinsed slo"l! to remo%e the cr!stal %iolet or &%e seconds# 6# Ne,t$ the smears "ere rinsed "ith the iodine reagent and then the same reagent "as applied or one minute# 5# The iodine reagent "as rinsed as in step three# :# The alcohol reagent "as applied slo"l!# The decolouri;er "as added until d!es does not run of rom the smears#
<# The alcohol "as rinsed immediatel! as in step three# =# The smears "ere co%ered "ith saranin reagent or 9> seconds# ?# It "as rinsed again and lotted dr! "ith a piece o lotting paper# 4># Each smear "as oser%ed under the oil)immersion o@ecti%e and the results "ere recorded# RESULTS
Bacillus
Staphylococcus aureus
8n(no"n +ulture
E.Coli 6>,
E.Coli 4>>,
8n(no"n A 6>,
8n(no"n A 4>>,
DISCUSSION
Bhat determines 'ram)positi%e and 'ram)negati%e acteria is due to diference in cell "all composition# Aecause the cell "alls o 'ram)negati%e cells ha%e a higher content o lipids and a thinner la!er o peptidogl!can$ the alcohol used in the decolori;ing step made the 'ram)negati%e cells incapale o retaining the meth!lene lue)iodine comple,# 3n the other hand$ 'ram)positi%e cells ha%e a thic(er peptidogl!can that traps the meth!lene lue)iodine comple,$ ma(ing it less %ulnerale to decolouri;ation# The 'ram stain technique "as used on un(no"n numer ># The un(no"n appeared to e 'ram negati%e and cocci shaped# Since this is an un(no"n$ the literature %alue is un(no"n# Possiilities o diference in
literature and e,perimental %alue could e due to e,cess primar! and mordant iodine d!es eing .ushed out entirel! ! the ?5C Ethanol$ or that the mordant iodine d!e "as not let on long enough#
CONCLUSION
'ram staining is used to determine the gram positi%e acteria "ill stain to a purple colour "hile the gram negati%e acteria stain to e in pin(ish colour#
QUESTIONS
4# /re there an! chemical diferences et"een the cell "all o gram positi%e and gram) negati%e acteria$ "hich might e,plain diferences in the rate o decolouri;ation The cell "all in acteria contains peptidogl!can$ a pol!mer o N)acet!l glucosamine$ N)acet!l muramic acid and amino acid# 'ram positi%e cell "alls contain a thic( la!er o peptidogl!can la!er that encircles the cells# Bhile the gram negati%e cell "alls contain a thin la!er o peptidogl!can et"een the c!toplasmicmemrane and the outer memrane# 'ram)negati%e acteria stained "ith cr!stal %iolet are decolori;ed ! ?5C alcohol "ithin min$ "hereas 'ram)positi%e acteria require at least 9 min treatment# /queous solutions o saranin$ neutral red$ and carol uchsine replace cr!stal %iolet rom stained 'ram) positi%e acteria more quic(l! than alcohol alone$ and alcoholic solutions o these counterstains are in most cases still more efecti%e# Treatment o cr!stal %iolet) stained organisms "ith alcoholic saranin or 45 sec "ill distinguish 'ram)positi%e acteria 1%iolet2 rom 'ram) negati%e acteria 1pin(2# /lcohol containing %er! lo" concentrations o iodine generall! decolori;es cr!stal %iolet)stained 'ram)positi%e acteria more quic(l! than •
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alcohol alone# Increasing concentrations o iodine in alcohol reduce the rate o decolori;ation o stained acteria$ ut stained 'ram)negati%e acteria are still readil! decolori;ed# The addition o iodine to alcohol increases the rate o e,traction o cr!stal %iolet ! alcohol rom 'ram)negati%e organisms$ ut dela!s e,traction o d!e rom 'ram)positi%e organisms$ and this applies "hen counterstain is also present# / t"o) solution modi&cation o 'ram staining is descried in "hich cr!stal %iolet)stained acteria are treated "ith an alcoholic solution o saranin$ carol uchsin$ and iodine# # 1a2 Does the age o the cultures efect the gram stain es 12 Bh! Aecause old culture o gram positi%e cells ma! not retain stain as "ell as !ounger cultures and could gi%e alse negati%e results# 9# 1a2 Aased on !our e,perience in the laorator! do !ou eel that the gram stain is a simple procedure I eel that the gram stain is not a simple procedure 12 Bh! For gram stain$ "e use more than one stain such as cr!stal %iolet$ iodine$ alcohol and saranin to our smears# 8nli(e the simple staining$ "e stained either cr!stal %iolet$ meth!lene lue or carol uchsine to our smears# Furthermore$ the cell "all o the acteria that react in gram stain has thic(er or thinner cell "all to (no" "hich acteria is a gram positi%e or gram negati%e# 6# Bhat is the in.uence o p0 on the gram stain reaction p0 afects the upta(e o cr!stal %iolet ! the acteria# +r!stal %iolet at lo" p0 and saranin do not stain the "alls o acteria# Bhen organisms o a gram positi%e are stained "ith cr!stal %iolet at a high p0 and then are "ashed at a lo" p0$ the d!e disappears rom the "alls# 3rganisms "ith stained "alls resist oth decolori;er and counterstain ater treatment "ith iodine and thus appear gram positi%e# •
