microlab gram stain

what is meningitis

a condition in which the protective membrane (meninges) around the brain and spinal cord become inflamed

meningitis causes damage to the

central nervous system

what is collected during a lumbar puncture

cerebrospinal fluid (CSF)

describe bacterial meningitis

medical emergency that requires prompt treatment with antibiotics

types of bacteria that cause meningitis

treptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae

describe gram stain

common technique that categorizes bacteria into gram positive and negative groups based on their cell wall structure

how long does gram staining take

a few minutes

what is the primary stain for this process

crystal violet

crystal violet is retained by

gram positive bacteria

gram positive have a ___ peptidoglycan layer

thick

safranin/pink is retained by which bacteria

gram negative

gram negative bacteria have a ___ peptidoglycan cell wall

thin

gram stains cannot identify

individual bacterial species within a group

what are the four reagents in gram staining

crystal violet

iodine

95% alcohol

safranin

describe crystal violet

stain both gram + and - bacteria purple by binding to negatively charged cell wall structures

CV molecules are + charged and bind to what

negatively charged bacterial cell wall structures

iodine is a

dye fixer (mordant)

iodine fixes the stain via

electrostatic forces, forming a large, insoluble crystal violet- iodine complex (CV-I)

95% alcohol

decolorizes gram - bacteria but not gram + due to the thicker peptidoglycan layer

safranin counterstains the gram - bacteria ___ while gram + bacteria remain ___

pink ; purple

what is the first step in gram staining

crystal violet 60s

second step in gram staining

iodine 60s

third step in gram staining

alcohol 5-10s

fourth step in gram staining

safranin 45s

what must be done after each step in gram staining

wash with water

gram positive bacteria consists of

cytoplasmic membrane, teichoic acids, and peptidoglycan layer

function of cytoplasmic membrane

surrounds cytoplasm, regulates passage of molecules in and out of the bacteria

teichoic acids function

protection from harmful substances, anchoring the peptidoglycan to the underlying plasma membrane, stabilizes cell wall

peptidoglycan function

provides rigidity and gives bactera it specific shape

what antibiotic inhibits the synthesis of peptidoglycan, causing the bacteria to die

penicillin

what does the gram negative cell wall consist of

cytoplasmic membrane, thin peptidoglycan layer (periplasmic space), outer membrane

describe the periplasmic space

lies between the outer and cytoplasmic membranes, containing a gel like matrix with vital proteins for cell functions

function of outer membrane

shields bacteria and hinders some antibiotics

porin proteins form

pores for hydrophilic molecule diffusion and lipopolysaccharides

lipopolysaccharides with a - charge function

helps maintain bacteria’s shape but can also cause toxic shock in infected hosts

describe bacterial smear

if the smear is applied in too thick of a layer, gram - bacteria appear darker and could be mistaken for gram +

describe fixation

if bacteria are over-heated during process, the cell wall is destroyed and bacteria are not capable of retaining crystal violet

describe expired culture smears

culture appears old, the bacteria; cell wall might have already broken down, rendering them incapable of retaining the crystal violet

describe under-decolorization

if alcohol (decolorizer) is washed away before it had any effect on the cell wall, the crystal violet will not escape the gram - cell wall

describe over decolorization

leaving alcohol on the slide for too long disrupts the cell wall of both gram + and gram -

gram positive bacteria is what control

positive ; staphylococcus is used

gram negative bacteria is what control

negative; escherichia is used

in a pure culture all bacteria are

identical

a mixed culture contains

different species of bacteria

human cells will appear reddish pink in gram stain because

it does not contain peptidoglycan