Lecture 1 Flashcards
Culture medium :
Nutrient material for growth of micoorganisms in the lab
Inoculum
Introducing microbes to media to initiate growth
Culture
Organisms that grow on culture media
Culture & sensitivity testing)
describe the test for culturing bacteria from patient specimen in hospital lab
Specimens arrive in lab in one of three forms -Swab, tissue, or fluid
AGAR
made from a red purple marine algae
* Complex water- soluble polysaccharide
* Solidifying agent for culture media
* Transparent & colorless
* Withstands the rigors of streaking
* Resists digestion by bacterial enzymes
* Inert, little effect on bacterial growth
* Solidifies at 30- 420C- common incubation temp for bacteria
* Melting temp is around 85C
* Remains liquid at >45C – so can add ingredients while still liquid and pour after sterilization and cooling
Final Concentration 0.05-0.1%
of MEDIA
WHAT IS CONSISTENCY
PURPOSE
APPLICATION
Liquid
* Slows down mixing of fluid
* Reduce convection currents
* Thioglycollate broth
0.2-0.5%
Final Concentration
of MEDIA
WHAT IS CONSISTENCY
PURPOSE
APPLICATION
Semi-solid
* Determine motility
* Prevents dispersion of acid/alkaline products
* Motility media
* O/F sugars
1 -2%
Final Concentration
of MEDIA
WHAT IS CONSISTENCY
PURPOSE
APPLICATION
Solid
* Solidification
* MacConkey
* Blood Agar
2-5%
Final Concentration
of MEDIA
WHAT IS CONSISTENCY
PURPOSE
APPLICATION
Solid
* Prevent swarming growth
* MacConkey
In order to use a type of culture media it must:
Be sterile
* passed QC testing
not expired
* Contain the right nutrients
* Have sufficient moisture
* Proper PH
* Be incubated at proper temp and atmosphere
* Oxygen or other gases must be available if required
Bacterial growth requirements
Water
* Sterile distilled or deionized water
* Peptones, meat infusions or meat extracts
Energy source
* Sugars, carbohydrates
Essential elements
* Basic building blocks of cells- carbon, nitrogen, sulfur, calcium, magnesium, potassium, iron, phosphorus)
Trace elements
* Sodium, zinc, molybdenum, cobalt, copper, etc..
Organic growth factors
* Blood, serum, yeast extract, vitamins, NAD
peptones
Proteins - a source of nitrogen & enzymes
* water-soluble form
* can withstand autoclaving
*made from proteins by hydrolysis
hydrolysates
Enzyme hydrolysis –enzyme is used to incorporate a water molecule between bonds to break up the protein
* break down meat or milk
* Tryptone is the best choice in media
Extract
Made by slow cooking a protein source until concentrated to a paste
Infusion
Made by leaving protein source in water for long periods of time until liquid infused with protein
* Both provide an extra source of vitamins and organic growth factors
* Are heat stable – add before autoclaving
* infusions made from calf brain or beef heart
Carbohydrates
Source of energy & cellular building blocks (e.g. carbon)
Used also to detect fermentation reactions
(with added indicator)
0.1-0.2% concentration - promote growth but dont alter pH
0.5-2.0%promote growth but alter pH - fermentation
5-10% - used in sugar utilization tests
Blood and blood products addition
added Trypticase soy agar or Columbia base agar
* fastidious org can grow
* Most bacteria grow healthier
* Cannot be autoclaved so blood added to after sterilization and cooling
* So must be collected from animal by aseptic technique
* And media must be checked for for sterility before use
* Blood used must be anticoagulated or defibrinated
Anticoagulated: chemical clot prevention
Defibrinated: mechanical fibrin removal – glass beads
agar with whole blood
Adding anticoagulated blood to media
* Provides essential nutrients
* 5- 10 % sheep or horse blood commonly used - bacteria may not look the same on both
* Human blood not recommended - may contain antibiotics or antibodies
* allows for visualization of whether bacteria can make a hemolysis enzyme that can break down the RBC
BA & CNA
Agar with Lysed blood
Red blood cells broken down by heating (850 C)
* Cellular components released – nutrients readily available for fastidious bacteria
- Chocolate Agar also has 2% hemoglobin & growth supplements
- Hemoglobin can be made by washing & autoclaving whole blood
*example Haemophilus influenzae ) - New York City agar (w antibiotics) – for isolating Neisseria gonorrhoeae
make Laked blood agar
Adding a lysing enzyme like pepsin or saponin to whole blood
-Hoyle’s medium (Corynebacterium diphtheriae)
* Brucella Kanamycin Vancomycin Laked Blood agar(KVLB) – used to isolate anaerobic gram negative
* Fildes agar- peptic digest of blood whole blood lysed by pepsin enzyme- for fastidious organisms (Haemophilus)
media with serum
-Serum is the fluid left over when whole blood clots
-serum = blood- fibrinogen
-source of electrolytes
* Enriches and helps solidify media
* Inspissation = heating of media & serum to 850 C so the proteins coagulate and solidify
* Horse serum most often used
Example: Loeffler’s medium for C. diphtheriae
Supplements added to media
selectivity for growth of a specific bacteria
* Or enhance growth of any bacteria
Vitamins
* Coenzymes
(example –Iso VitaleX)
Example : Thioglycollate broth
media with pH Indicators:
acidity or alkalinity detected by a color change
* dye that acts as a pH indicator is added
* Neutral red, Phenol red, ,Bromthymol blue
Redox or Eh Indicator:
* detect presence or absence of oxygen.
* Used in Thioglycollate broth
* Example of a redox indicator in media: Resazurin
Selective agents in media
-select for some bacteria but inhibit others
-heat stable add before autoclaving
Bile salt mixtures -inhibit gram positive organisms
Desoxycholate -inhibit gram positive organisms Dyes
* Crystal violet (inhibit gram positive organisms)
* Brilliant green (inhibit gram positive and some negative organisms)
Salts - In high concentrations is a selective agent.
antibiotics as selective media
very heat sensitive
* prepare aseptically must be added to media after autoclaved and cooled
CNA:
* has Colistin & Nalidixic acid which allows gram positive organisms to grow while preventing the growth of most gram negative
NYC:
* has numerous antibiotics to try and grow only N. gonorrhoea – See later slide for specific antibiotics and which organisms are killed
Buffers
Control pH fluctuations in media due to bacterial metabolism
* Peptones
* Inorganic phosphates: buffer and serve as a source of phosphorous
Charcoal/Starches
Absorb toxic substances
* inhibit the growth of fastidious organisms
-Transport media, BCYE (Buffered Charcoal Yeast Extract agar)
Basic media
Simple media, only barest amount of nutrients water, agar, simple proteins
Fastidious organisms will not grow
No differential or selective properties
Trypticase soy Agar
* Nutrient Agar
* Brain Heart Infusion Agar
* Columbia Agar Base
* Mueller Hinton Agar
Enriched media:
-Basic media + extra nutrients like vitamins or blood
-usually a broth
-Brilliant green, selenium salts (feces) or tetrathionate suppress growth of normal flora for a short period of time
Selenite broth, Tetrathionate broth, Brain
Heart Infusion broth, Cooked meat broth
Differential media:
Has ingredients that allow you to tell one bacteria from another when you have mixed bacteria growing.
Mueller Hinton Agar
A Basic media for susceptibility testing (AST) of non fastidious organisms
* Has proteins in the form of extracts & enzyme hydrolysates
* Has starch to absorb toxins ,helps with antibiotic diffusion & has dextrose for energy
* Agar is loose for better diffusion of the antibiotics
* Shows batch-to-batch reproducibility for AST testing
* Is low in sulfonamide, trimethoprim, and tetracycline inhibitors like thymidine and thymine
can add sheeps blood to make more selective
Chocolate agar
Enriched because has 5-10% Sheep blood that has
been hemolysed by heating to 800C or by adding an enzyme – agar looks brown like chocolate because has
2% hemoglobin + supplements
* Grows most organisms but used especially, to grow more fastidious microorganisms
* Not a differential media because you can’t see whether hemolysin enzyme produced by bacteria- blood already hemolysed
you cannot see homolysis on this plate because the blood is already hemolysed
Thioglycollate broth
-General-purpose, enriched, differential media
-Enriched with hemin and vitamin K to grow fastidious & anaerobes.
* Differential because you can see O2 requirements of what is growing
- Some have redox resazurin dye to monitor levels of O2 – turns pink in O2
when growing aerobes the colonies will be at the top - highest oxygen
anaerobic - at the bottom of the test tube
facaltative anaerobes - everywhere but concentrated on the top
Colistin Naladixic Agar
CNA
Enriched because has 5-10 % whole sheep blood
* Selective because has antibiotics
Colistin and Naladixic acid that will inhibit gram neg growth
* Allows gram positive organisms and yeast to grow
* Differential because allows you to see which organisms can hemolyze RBC
Macconkey Agar
selective Ingredients:
* Bile salts
* Crystal violet
* Eliminate Gram positive & grows gram negative
Differential ingredients:
* Lactose (carbohydrate)
* Neutral Red (pH indicator)
* Lactose fermenters (LF) – bright pink colonies
* Non-lactose fermenters (NLF) – colourless colonies
mannitol salt agar
Selective differential agar for Staphylococcus aureus
Selective ingredients:
* A high concentration of salt 7.5% (halophilic)
Differential ingredients:
* pH Indicator: Phenol red (yellow in acid, pink/red in basic)
* Carbohydrate: Mannitol
* Differentiate between pathogenic and nonpathogenic Staphylococcus
* S. aureus grow and ferment mannitol – yellow
* Other Staph species grow but do not ferment mannitol – pink
Sorbitol Macconkey SMAC
Selective and differential media
* Selective for gram negative – inhibitory to gram positives
* Same ingredients as MAC except sorbitol instead of lactose
* Differentiates between entero-hemorrhagic Escherichia coli (EHEC) which fail to ferment sorbitol and Escherichia coli which will ferment sorbitol and do not cause diarrhea
Sorbitol Fermenters = PINK
Salmonella SHIGELLA SS
Selective & differential agar for enteric (intestinal) pathogens
Selective ingredients:
* Bile salts + sodium citrate + brilliant green
* Inhibits all gram-positive & gram-negative that are normal flora
* Highly selective for enteric pathogens - that cause food poisoning
Differential ingredients:
* Lactose and neutral red (pH indicator)
* Differentiate between LF and NLF
* Sodium thiosulphate + ferric ammonium citrate
* Differentiate between H2S and non-H2S producing colonies
hydrogen sulfide production in media
▪ hydrogen sulfide reacts with ferric ions from the ferric ammonium citrate to make an insoluble heavy metal black precipitate
▪ H2S positive result is seen as a blackening of the center or entire colony
LF, H2S positive Pink colonies with black centres
NLF, H2S positive Clear colonies with black centres
NLF H2S negative Clear colonies
Hektoen Agar
Selective and differential agar used to isolate enteric pathogens
Selective ingredients:
* Bile salts inhibit gram positive organisms
Differential ingredients:
* sugars- lactose, sucrose and salicin
* Indicator: Bromthymol blue + acid fuchsin
* Na thiosulfate and ferric ammonium citrate in the medium produces a black precipitate in the presence of H2S
fermenters that are H2S postive - orange/yellow colonies with black center
non fermenters with h2s postive - green with black centers
fermenters with NO H2S -orange/yellow
non fermenter with no h2s blue green colonies
Cefsulodin irgasan novobiocin - CIN agar
Selective and differential media specifically used to isolate the enteric pathogen Yersinia enterocolitica
Selective ingredients
* Cefsulodin, Irgasan, Novobiocin, Na desoxycholate & crystal violet all inhibit gram positive organisms
* Also inhibit gram negative that are normal flora in stool
Differential ingredients
* Sugar = Mannitol & Neutral red pH indicator
* mannitol fermenters -pink with clear edges
* Look like a bulls eye
Campy agar
Selective enriched media for Campylobacter – is microaerophilic
Enriched ingredients:
* 10% sheep blood
Selective ingredients:
* Vancomycin inhibit gram positive organisms
* Amphotericin B & Polymyxin B inhibit yeast/fungus
* Trimethoprim inhibits a swarming gram negative called Proteus
* Cefoperazone inhibits gram negative organisms
* Na bisulfite lowers the O2 levels enhancing the recovery of microaerophilic organisms
new york city agar
Selective enriched media used specifically for the isolation of Neisseria gonorrhoea and Neisseria meningitidis
Enriched ingredients:
* Has hemoglobin from lysed horse blood, yeast dialysate & horse plasma
Selective ingredients:
* Vancomycin inhibits gram positive
* Colistin inhibits gram negative
* Amphotericin B inhibits yeast
* Trimethoprim inhibits swarming Proteus
chromogenic agar
has soluble colourless molecules called chromogens
* Chromogens consist of a substrate and a chromophore
* If the target organism is present the specific enzyme it makes cleaves the colourless chromogenic conjugate, releasing the chromophore
* When the chromophore is not in the conjugated form (unconjugated) it exhibits it’s distinct color which precipitates
on the colony
advantages - Provides a color based differentiation
* Clearly distinguishable
* Colonies of specific microorganisms can be recognized by their colour
quality control of media
Expiry date, lot numbers
* Physical appearance: depth of agar (4mm), cracks, excessive bubbles, contamination
* Performance: test with known ATCC strains to ensure proper growth and appearance
BETA HEMOLYSIS
RBC are destroyed by exotoxins or enzymes produced by the bacteria as they grow on media
only report on (BA, CNA) NOT choc
* Causes complete breakdown or lysing of RBC cell membrane
* Seen as a clear zone (media becomes see through) around and under the colony
Some bacteria produce multiple toxins or enzymes and have larger zones of beta hemolysis
* Some produce haemolysins that are oxygen labile-only active in decreased O2
Clostridium perfringens is a strict anaerobic** organism Called double zone beta hemolysis Theta & alpha toxin
staph aurea
ALPHA HEMOLYSIS (AH or α H)
partial breakdown of RBC
* Bacteria produce hydrogen peroxide which oxidizes hemoglobin to methemoglobin
* Result is a green zone in the media around and under colony
BUT THE COLONIES ARE GREY
* Seen more intensely on the CNA plate
- Streptococcus pneumoniae
Umbilicate
innie”, “like a checker piece” ^-^
* Streptococcus pneumoniae
* tilt plates to examine
Umbonate
“outie”
Viridans streptococcus ^
ways to report size
pinpoint, small , medium and large
Most common colors used to describe bacteria
white
yellow
tan
grey
red
METALLIC SHEEN
Pseudomonas (a strict aerobe),
is a NLF gram negative
organism only for this organism
Quantity
Scant
Light
Moderate
Heavy
depending on quadrant
GRAM STAIN
based on cell wall structural differences of GN GP
infections caused by a single organism = monomicrobial
* by multiple organisms = polymicrobial
Gram positive cell wall
Thick, peptidoglycan layer (90%)
* Many teichoic acid cross-linkages
* Lower lipid content , 0-2%
* No periplasmic space
* Resists decolourization
Gram negative cell wall:
Thin peptidoglycan layer (5-20%)
* No teichoic acid cross-linkages
* Higher lipid content 10-20%
* Have both an outer and inner membrane
* Has periplasmic space
* Does not resist decolourization
CRYSTAL VIOLET- PRIMARY STAIN
Basic stain so it binds to the negatively charged groups in the cell with ionic bonds (po4)
* Diffuses through cell wall pores & bonds with negatively charged cell groups (e.g. phosphate groups in nucleic acid)
-water does not decolorize
IODINE (MORDANT)
Diffuse into the cell
* Combines with crystal violet forming crystal violet-Iodine (CVI) complex
* Traps crystal violet making it hard for it to leave the cell
* CVI complex is stable in water but soluble to solvents such as alcohol & acetone
TRAPPING AGENT
ACETONE ALCOHOL (DECOLORIZER)
Most important step …. Timing is critical
* ethanol and acetone
* Extracts lipid creating larger pores in cell wall makes it more permeable
* Dehydrates peptidoglycan reducing pore size & movement
In GN cell wall:
* high lipid, low peptidoglycan
* Lipids extracted and CVI complex leaves cell
In GP cell wall:
*low lipid, high peptidoglycan
* Peptidoglycan dehydrated and CVI complex trapped
Looking under the microscope at this point: GN will look
colorless and GP will appear bluish purple
SAFRANIN COUNTERSTAIN
can also use dilute Carbolfuchsin
* No effect on GP as binding sites were blocked by CVI complex
* Forms bonds with cell groups in GN cell wall
* GP are bluish purple and GN are pink
general rules of aseptic techniques
Keep petri dishes closed when possible
Clean and disinfect lab surfaces prior to and after use
Avoiding talking/breathing on cultures
Use a face shield when procedures create aerosols
Wash your hands & wear gloves & lab coat
Open packages in a manner where you do not touch the parts that will come in contact with your sterile media
Sterilize inoculating loops and other equipment that comes into contact with cultures
gram positive color
white(staph) gold/yellow staph aur , grey - shiny on the edges - strep
smaller colonies - CNA
the smallest bacteria we see very pinpoint in a slide
coccobacilli - negative
gram postive bacilli
always either large or small (small chinese letter paliside)
diplococci
football or cat eye shape in gram pos only
cant have gram neg
why is gram stain important in csf
to determine the antibiotic type
can be used to differentiate and quntitate inflammatory and epithelial cells
first test in culture test
cells are naturally
transparent
gram stain is a
differntial stain
because it uses two dyes and seperates two types of bacteria
if there is under decolorization
decolorization time was too short
gram negs appear blue
if there is over decolorization
decolorization time too long
gram pos are red
can happen if the cells are too old
