Microbio Notes

8/12/2019 Microbio Notes

http://slidepdf.com/reader/full/microbio-notes 1/13

Coverage of LT – Chapter 5, 6, 12, 13

Chapter 6

Bacterial Growth

• Estimating the nm!er of cells !" !inar" fission #ring growth phase

• E$presse# as the nm!er of via!le %living& organisms per mL of cltre

'erial (iltion an# 'tan#ar# plate cont

• Loo) for a pictre* #iltion continosl"

• Base# on the fact that n#er normal living con#itions, onl" via!le+ living !acterim will

#ivi#e an# form a visi!le colon" on an agar plate

• Becase it is #ifficlt to cont more than 3 colonies, it is necessar" to #ilte the

original cltre !efore "o transfer a )nown volme on an agar plate

• Each !acterim represents a colon" forming nit %C-.&

• /roper serial #iltion "iel#s conta!le nm!er of colonies %3 to 3 per plate&

'prea#0plate metho#

• 'ample is pipette nto srface of agar plate

• 'ample is sprea# evenl" over srface of agar sing sterile glass sprea#er

• nc!ation

• 'rface colonies

/or0plate metho#

• 'ample is pipette into sterile plate

• 'terile me#im is a##e# an# mi$e# well with inoclms

• nc!ation

• '!srface an# 'rface colonies

o ot that sefl in antif"ing colonies

4ia!le /late Cont

• To cont the nm!er of colonies, the plate mst !e viewe# n#er the magnif"ing lens of



a colon" conter

ccrac" of 'erial (iltion an# /late Cont etho#

• (epen#s on homogenos #ispersal of cells in each #iltion

• 'ha)e each cltre !efore sampling

• a)e several plates %triplicates& from each #iltion

• (oes not incl#e cells that ma" have #ie# #ring plating %colonies represent the nm!er

of living cells&

(irect microscopic conts

• Bacterial sspension is place# on sli#e an# seeps n#er cover slip7 'spension fills

shallow space of )nown volme over gri#8

ost /ro!a!le m!er

• .se# when samples contain too few cells to give relia!le measres of poplation si9e !"

the stan#ar# plate cont or cells will not grow on agar plates

• ore alitative than antitative

• 4isi!le inspection of certain meta!olic pro#cts:

Tr!i#it" %spectrophotometric or colorimetric&

• easres !acterial growth !" #etermining the optical #ensit" %;(& of the cltre

• ot goo# for samples with high cell #ensit" %nee# #iltion&, cannot #istingish #ea# cell

particles from via!le cells

(r" weight

• Centrifge cells an# collect the pellet an# free9e #r" to "iel# pow#ere# samples %mg+L7

g+L, etc7&

tritional -actors ffecting Bacterial Growth

• acrontrients – reire# in large amonts

• icrontrients – reire# in lesser amonts

• -asti#ios – microorganisms that have special ntritional nee#s that ma" !e #ifficlt to

!e provi#e# in the la!orator"

trient -orm of trient in the -orm spplie# in cltre



Environment me#ia

Car!on C;2, organic compon#s Glcose,malate, acetate,p"rvate, amino aci#s orcomple$ mi$tres li)e "easte$tract an# peptone

itrogen <3, ;3, 2, organic

compon#s

norganic= <>Cl,

%<>&2';>, 2

;rganic= mino aci#s,nitrogen !ase of ncleoti#es

/hosphors

tritional Comple$it"

• icroorganisms with man" en9"mes have simple ntritional nee#s !ecase the" can

s"nthesi9e nearl" all the s!stances the" nee#7 Those with fewer en9"mes have

comple$ ntritional reirements !ecase the" lac) the a!ilit" to s"nthesi9e the

s!stances the" nee# for growth7

Location of En9"mes

• E$tracelllar %e$oen9"mes& – sall" pro#ce# !" Gram ? ro#s for splitting large

molecles of car!oh"#rate, lipi# or protein in the me#im

• /eriplasmic an# c"toplasmic %en#oen9"mes&

#aptation to Limite# trients

• '"nthesis of increase# amonts of en9"mes7 ;!tains an# ses larger proportions of the

few ntrients availa!le7

• '"nthesi9e en9"mes to o!tain a more plentifl ntrient availa!le7

• #@stment of the rate at which ntrients are meta!oli9e# an# the rate at which

molecles are s"nthesi9e# to fit the availa!ilit" of the limite# ntrient

• -ormation of en#ospore or sporlation7 Aesistant to high temperatres, ra#iation an#

to$ic chemicals %Clostridium an# Bacillus& -orme# as c"sts !" protists li)e Entamoebaspp7 n# Trichomonas vaginalis.

'porlation

• En#ospores

• ;ther spore0li)e !acterial creatres



• -orme# when there are limite# ntrients

etho#s of ;!taining /re Cltres of Bacteria

• /re Cltre

o cltre that contains onl" a single species of organism7

o mportant in st#"ing !acteria in the la!orator"7

o ;!taine# from strea)s or highest #iltion of a certain sample7

septic Technies

• inimi9es the chances that pre cltres will !e contaminate# !" organisms from the

environment or that organisms, especiall" pathogens, will escape into the environment7

'trea) /late etho#

• Bacteria are pic)e# p !" a wire loop an# strea)e# on agar srface

• The loop is flame# an# pic)0p !acteria from the previos strea)s to ma)e new strea)s

• Aepeate# strea)ing thins ot the concentration of cells from the first strea)7

• Colonies appear after inc!ation at a sita!le growth temperatre

• The loop is se# to pic) p in#ivi#al colonies an# strea)e# again in plates again or

inoclate# in lii# me#im

/or /late etho#

Cltre e#ia

• atral e#ia

o n natre, marine, freshwater, terrestrial ha!itats

o i$e# organisms

• (efine# '"nthetic e#im

o /repare# in the la!orator"

o Contains )nown specific )in#s an# amonts of chemical s!stances

• Comple$ e#im

o /repare# in the la!orator" !t chemicall" non#efine#



Commonl" .se# me#ia in soli# or lii# form contain=

• /eptone0 provi#es pepti#es that micro!es can se

• east e$tract – provi#es vitamins, coen9"mes an# ncleosi#es

• Casein h"#rol"sate – from mil), provi#es amino aci#s

• 'erm or whole !loo# – contains man" ntrients for pathogens7 %E$7 Bloo# agar – sefl

in i#entif"ing pathogens which !rea) #own re# !loo# cells or hemol"sis7&

'elective me#im – encorages growth of some organisms !t sppresses the growth of others

(ifferential me#im – has a constitent that case an o!serva!le change %color or p< change&

in the me#im when a particlar !iochemical reaction occrs7

some me#ia can !e !oth selective an# #ifferential

E$ample=

• acCon)e" agar – cr"stal violet an# !ile salts allow growth of Gram – onl"

o Lactose an# p< in#icator 0D will trn colonies of lactose fermenters to re#* non0

fermenters colorless an# translcent

• Enrichment me#im

o Contains special ntrients that allow growth of a particlar organism that might

not otherwise !e present in sfficient nm!ers to allow it to !e isolate# an#

i#entifie#7

o E$amples= selenim a##e# to me#im to enrich growth of Salmonella typhi from

fecal sample

aintaining Cltres

• 'toc) cltre – is never se# for la!orator" st#ies* when nee#e# for st#ies, it is

inoclate# into a fresh me#im7

o re maintaine# !" ma)ing s!cltres in fresh me#im reglar intervals to )eep

the organism growing

• /reserve# cltre – a cltre )ept in a #ormant stage to avoi# the ris) of contamination

an# to re#ce mtation rate

o ost common se# technie= free9e0#r"ing or l"ophilisation

• Aeference cltre – a preserve# cltre that maintains the organisms with the

characteristics as originall" #efine#



o aintaine# at merican T"pe Cltre Collection %TTC& an# man" also

maintaine# in niversities an# research centres

etho#s of ltiple (iagnostic Tests

• Enterot!e mlti test s"stem

o llows simltaneos #etermination of an organism8s reaction to a variet" of

#iagnostic me#ia from a single inoclation

o #entifies enteric pathogens that case intestinal #iseases li)e t"phoi# fevers,

sastroenteritis an# foo# poisoning7

o Consists of compartments, inoclate# an# inc!ate# at 3C for 2> hors

o Test are grope# in 38s assigne# as 1,2 an# >

o

The sm of the nm!ers of positive tests in each grop in#icates which tests arepositive

o list of i#entification nm!ers an# the correspon#ing microorganism is provi#e#7

• nal"tical /rofile n#e$ %/&

o 'imilar to Enterot!e '"stem !t me#ia tra"s7

Chapter 12

'terili9ation an# (isinfection

'terilit" –means that there are no living organisms in or on a material

• <eat 'terili9ation

o <eat is the most wi#el" se# metho# of sterili9ation

o ;ften, however, we cannot attain sterilit", !t we can still control microorganisms

effectivel" !" limiting their growth, throgh the process of inhi!ition7

o The temperatre mst eliminate the most heat0resistant organisms, sall"

!acterial en#ospores7

o <eat rapi#l" penetrates thic) materials nli)e chemicals

• (r" <eat

o -or metal o!@ects an# glasswares

o 11 C for 1 h* 16 C for 2 h or longer* 121 C for 26 h or longer %#epen#ing on⁰ ⁰ ⁰



volme&

o ;pen -lame – e$7 lcohol lamp or Bnsen !rner

-or inoclating loops, moth of glass wares an# insi#e of glass pipettes

• oist <eat

o toclave – seale# heating #evise that allows the entrance of steam n#er

pressre

o ain action is protein #enatration an# ma" #isrpt mem!rane lipi#s as well

o /rions are highl" resistant to atoclaving 0 D shol# !e 13> C, 1F mintes⁰

o t !oiling point, 1 c, water #estro"s vegetative cells of most !acteria an# fngi⁰

an# inactivates virses !t cannot )ill all )in#s of spores7

o f water is heate# n#er pressre its !oiling point is elevate#, so temperatres

a!ove 1 c can !e reache#7 This is accomplishe# !" sing an atoclave7⁰

o /ressre is set at 15 l!+in2 maintaine# at 15 to 2 mintes at this pressre the

heat reaches 121 C enogh to )ill spores an# ncleic aci#s of virses⁰

• /asteri9ation

o nvente# !" Lois /aster

o (oes not sterili9e lii#s !t re#ces micro!ial loa#, )illing most pathogens an#

inhi!iting the growth of spoilage microorganisms7

o ills Samonella an# Mycobaterium

o ll evaporate# or con#ense# canne# mil) is sterile, these are sterili9e# !" steam

n#er pressre hence the coo)e# flavor nli)e fresh mil) la!elle# Hpasteri9e#8

o il) sol# in tetra pac)s are pasteri9e# in higher temperatres

• Aefrigeration, free9ing, #r"ing an# free9e0#r"ing

o Col# temperatre retar#s the growth of microorganisms !" slowing the rate of

en9"me0controlle# reactions !t #oes not )ill man" micro!es7 <eat is mch more

effective in )illing microorganisms7

• Aefrieration

o To prevent spoilage !" slowing #own the rate of chemical reactions at 5 c⁰

o n rare instances, strains of Clostridium botulinum were fon# growing in the



refrigerator where anaero!ic con#itions persists

• -ree9ing

o 'torage of foo# at 02 c is se# for preservation in homes an# in foo# in#stries⁰

o lso for preservation of microorganisms !t lower than 02 C7 icroorganisms⁰

are sall" sspen#e# in gl"cerol to prevent the formation of ice cr"stals which

col# pnctre cells coole# with soli# car!on #io$i#e %#r" ice& to temperatre of

0F C7⁰

o Lii# nitrogen – mammalian cells at 01F C⁰

• (r"ing

o !sence of water inhi!its the action of en9"mes

o inimi9es also the sprea# of infectios agents

E7g7 #r"ing of lan#r" in #r"ers or n#er the sn

o (rie# an# smo)e meat an# fish retain moistre for microorganisms to grow7

'ealing them in plastics creates an anaero!ic con#ition for Clostridium botulinum

to grow7

o Treponema palladium which cases s"philis is e$tremel" sensitive to #r"ing an#

#ie almost instantl" on #r" srfaces7 Ths it can !e prevente# from sprea#ing !"

)eeping toilet seats an# other !athroom fi$tres #r"7

• Aa#iation

o Controlle# #oses of electromagnetic ra#iation effectivel" inhi!it micro!ial growth7

o icrowaves

o .4 ra#iation %2203 nm an# near0visi!le .4&

o oni9ing ra#iation %gamma ra", I0ra"&

o .4 light is of limite# se !ecase it #oes not penetrate glass, cloth, paper or

most other materials an# it #oes not go aron# corners or n#er la! !enches

o t penetrates air, effectivel" re#cing the nm!er of air!orne microorganisms

)illing them on srfaces of operating rooms, cltre rooms, etc7

o 'ewage efflents are rn n#er .4 light in some .' commnities to #estro"

microorganisms as replacement for chlorine which is more costl"7

• 'onic an# .ltrasonic Javes



o 'onication

(isintegration of cells throgh son# waves

• -ilter 'terili9ation

o -ilter – a #evice with pores too small for the passage of microorganisms !t large

enogh to allow passage of lii#+ gas

o <igh efficienc" particlate air filter

Chapter 13

ntimicro!ial Therap"

• Chemotherap"

o Coine# !" /al Ehrlich

o .se of chemical s!stances to )ill pathogens withot in@ring the host

o To#a", refers to se of chemical s!stances to treat varios aspects of #isease

o (rgs or chemotherapetic agents – an" chemical s!stance se# in me#icinal

practice

• ntimicro!ial gents

o atral or s"nthetic chemical that )ills or inhi!its the growth of microorganisms

Ci#al agents – )ill organisms

'tatic agents – #o not )ill !t onl" inhi!it growth

• inimm nhi!itor" Concentration %C&

o The effectiveness of an anti!acterial chemical agents is assesse# !" #etermining

the minimm concentration necessar" to completel" inhi!it !acterial growth

• (etermining icro!ial 'ensitivities

o (is) (iffsion etho#+ ir!"0Ba"er etho#

trient agar

'prea# plate microorganism

nti!iotic in each #iscs place# on the agar srface



nc!ate

Test organisms shows sensitivit" to some anti!iotics, in#icate# !"

inhi!ition of !acterial growth aron# #iscs

o (iltion etho#

o 'erm

• inimm Bacterici#al Concentration

o The lowest concentration of the chemotherapetic agent "iel#s no growth

following the inoclation of non0tr!i# #iltions

o This concentration mst !e maintaine# at the site of infection !ecase it is the

minimm amont that will cre the #isease

•'erm illing /ower

o ;!tain a sample of patients8 !loo# while patient is receiving an anti!iotic

o !acterial sspension is a##e# to a )nown antit" of the patient8s serm

o ;!serve for a!sence of growth7 f tr!i#it" is seen ti means anti!iotic is

ineffective

• tomate# metho#s

o a)e la!orator" i#entification of organisms an# their sensitivities to

antimicro!ials more efficient an# less effective

• ntimicro!ial gents se# in 44;

o ttri!tes of an #eal nitmicro!ial gent

'elective to$icit"

'ol!ilit" in !o#" fli#s

To$icit" not easil" generate#

on0allergenic

'ta!ilit"= maintenance of a constant, therapetic concentration in !loo#

an# tisse fli#s

Long shelf0life an# reasona!le cost

Aesistance !" microorganisms not easil" acire#



• o#es of ction of ntimicro!ials

o nhi!ition of cell wall s"nthesis

o (isrption of cell mem!rane fnction

o nhi!ition of protein s"nthesis

o nhi!ition of cleic ci# '"nthesis

o ntimeta!olites %moleclar mimicr"&

• nti!acterials – antimaeta!olits

o '!stances that affect the tili9ation of meata!olites an# ths prevent a cell from

carr"ing ot necessar" meta!olic reactions

o 'trctrall" similar to normal meta!olites

o Kmoleclar mimicr"

o 'lfnami#es

o sonia9i#

o Etham!tol

o 4i#ara!ine

o

#$ri#ine

• 'i#e effects

o To$icit"

o llergies

• ntifngals

• ntiviral

o nhi!it a phase of a virs !t #oes not )ill it

• Aesistance to (rgs

o eans that a microorganism formerl" sscepti!le to the action of the anti!iotic is

no longer affecte# !" it

o <ow is it acire#



Evasion – non0genetic resistance

Genetic resistance #e to=

• Chromosomal changes – sall" effective onl" against a single

t"pe of anti!iotic

• /lasmi#e !orne or e$trachromosomal resistance – #e to

presence of A plasmi#s or A factors

o ;rigini of A plasmi#s0 the" e$iste# in the natral micro!ial

poplation even !efore anti!iotics were #iscovere# or se#

• echanisms of (rg Aesistance

o tation in Target molecles

tation alters the ( so protein pro#ce# is mo#ifie# an# can8t !in# tothe target

• Aesistance to er"throm"cin, rifam"cin an# antimeta!olites

lterations in mem!rane permea!ilit"

• ew genetic information changes the natre of proteins in the

mem!rane

• E7g7 Aesistance to tetrac"clines, inolones an# some

aminogl"cosi#es

En9"me #evelopment

• common case of resistance* can #estro" or inactivate

antimicro!ial agents

o E7g7 !eta0lactamases capa!le of !rea)ing the !eta0lactam

ring in penicillins an# some cephalosporins

En9"me activit" changes

• This mechanism allows a formerl" inhi!ite# reaction to occr

o E7g slphonami#e – resistant !acteria have #evelope# an

en9"me that has a ver" high affinit" for /B an# ver" low

affinit" for slphonami#e

lterations in na!olic /athwa"s



• B"passes a reaction inhi!ite# !" an antimicro!ial agent

o E7g7 in other slphonami#e0resistant !acteria that se

rea#"0ma#e folic aci# from their envirionment

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