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Micro > Lecture 1 > Flashcards

Lecture 1 Flashcards

1
Q

Culture medium :

A

Nutrient material for growth of micoorganisms in the lab

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2
Q

Inoculum

A

Introducing microbes to media to initiate growth

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3
Q

Culture

A

Organisms that grow on culture media

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4
Q

Culture & sensitivity testing)

A

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

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5
Q

AGAR

A

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

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6
Q

Final Concentration 0.05-0.1%
of MEDIA
WHAT IS CONSISTENCY
PURPOSE
APPLICATION

A

Liquid
* Slows down mixing of fluid
* Reduce convection currents
* Thioglycollate broth

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7
Q

0.2-0.5%
Final Concentration
of MEDIA
WHAT IS CONSISTENCY
PURPOSE
APPLICATION

A

Semi-solid
* Determine motility
* Prevents dispersion of acid/alkaline products
* Motility media
* O/F sugars

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8
Q

1 -2%
Final Concentration
of MEDIA
WHAT IS CONSISTENCY
PURPOSE
APPLICATION

A

Solid
* Solidification
* MacConkey
* Blood Agar

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9
Q

2-5%
Final Concentration
of MEDIA
WHAT IS CONSISTENCY
PURPOSE
APPLICATION

A

Solid
* Prevent swarming growth
* MacConkey

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10
Q

In order to use a type of culture media it must:

A

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

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11
Q

Bacterial growth requirements

A

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

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12
Q

peptones

A

Proteins - a source of nitrogen & enzymes
* water-soluble form
* can withstand autoclaving
*made from proteins by hydrolysis

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13
Q

hydrolysates

A

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

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14
Q

Extract

A

Made by slow cooking a protein source until concentrated to a paste

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15
Q

Infusion

A

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

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16
Q

Carbohydrates

A

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

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17
Q

Blood and blood products addition

A

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

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18
Q

agar with whole blood

A

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

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19
Q

Agar with Lysed blood

A

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
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20
Q

make Laked blood agar

A

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)

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21
Q

media with serum

A

-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

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22
Q

Supplements added to media

A

selectivity for growth of a specific bacteria
* Or enhance growth of any bacteria
Vitamins
* Coenzymes
(example –Iso VitaleX)
Example : Thioglycollate broth

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23
Q

media with pH Indicators:

A

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

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24
Q

Selective agents in media

A

-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.

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25
Q

antibiotics as selective media

A

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

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26
Q

Buffers

A

Control pH fluctuations in media due to bacterial metabolism
* Peptones
* Inorganic phosphates: buffer and serve as a source of phosphorous

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27
Q

Charcoal/Starches

A

Absorb toxic substances
* inhibit the growth of fastidious organisms
-Transport media, BCYE (Buffered Charcoal Yeast Extract agar)

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28
Q

Basic media

A

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

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29
Q

Enriched media:

A

-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

30
Q

Differential media:

A

Has ingredients that allow you to tell one bacteria from another when you have mixed bacteria growing.

31
Q

Mueller Hinton Agar

A

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

32
Q

Chocolate agar

A

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

33
Q

Thioglycollate broth

A

-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

34
Q

Colistin Naladixic Agar

A

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

35
Q

Macconkey Agar

A

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

36
Q

mannitol salt agar

A

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

37
Q

Sorbitol Macconkey SMAC

A

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

38
Q

Salmonella SHIGELLA SS

A

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

39
Q

hydrogen sulfide production in media

A

▪ 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

40
Q

Hektoen Agar

A

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

41
Q

Cefsulodin irgasan novobiocin - CIN agar

A

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

42
Q

Campy agar

A

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

43
Q

new york city agar

A

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

44
Q

chromogenic agar

A

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

45
Q

quality control of media

A

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

46
Q

BETA HEMOLYSIS

A

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

47
Q

ALPHA HEMOLYSIS (AH or α H)

A

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
48
Q

Umbilicate

A

innie”, “like a checker piece” ^-^
* Streptococcus pneumoniae
* tilt plates to examine

49
Q

Umbonate

A

“outie”
Viridans streptococcus ^

50
Q

ways to report size

A

pinpoint, small , medium and large

51
Q

Most common colors used to describe bacteria

A

white
yellow
tan
grey
red

52
Q

METALLIC SHEEN

A

Pseudomonas (a strict aerobe),
is a NLF gram negative
organism only for this organism

53
Q

Quantity

A

Scant
Light
Moderate
Heavy
depending on quadrant

54
Q

GRAM STAIN

A

based on cell wall structural differences of GN GP

infections caused by a single organism = monomicrobial
* by multiple organisms = polymicrobial

55
Q

Gram positive cell wall

A

Thick, peptidoglycan layer (90%)
* Many teichoic acid cross-linkages
* Lower lipid content , 0-2%
* No periplasmic space
* Resists decolourization

56
Q

Gram negative cell wall:

A

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

57
Q

CRYSTAL VIOLET- PRIMARY STAIN

A

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

58
Q

IODINE (MORDANT)

A

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

59
Q

ACETONE ALCOHOL (DECOLORIZER)

A

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

60
Q

SAFRANIN COUNTERSTAIN

A

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

61
Q

general rules of aseptic techniques

A

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

62
Q

gram positive color

A

white(staph) gold/yellow staph aur , grey - shiny on the edges - strep
smaller colonies - CNA

63
Q

the smallest bacteria we see very pinpoint in a slide

A

coccobacilli - negative

64
Q

gram postive bacilli

A

always either large or small (small chinese letter paliside)

65
Q

diplococci

A

football or cat eye shape in gram pos only

cant have gram neg

66
Q

why is gram stain important in csf

A

to determine the antibiotic type
can be used to differentiate and quntitate inflammatory and epithelial cells

first test in culture test

67
Q

cells are naturally

A

transparent

68
Q

gram stain is a

A

differntial stain
because it uses two dyes and seperates two types of bacteria

69
Q

if there is under decolorization

A

decolorization time was too short
gram negs appear blue

70
Q

if there is over decolorization

A

decolorization time too long
gram pos are red

can happen if the cells are too old

Micro (15 decks)

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