Microbiology Lab Practical – Flashcards

Microscope occular. 

Eye peice (usually 10x magnification)

Microscope objective

we use 100x for oil immersion

Microscope condensor

directs light towards the objective lens in bright feild micsopcopy

resolving power.

expressed as d (the smallest distance between two objects that can be seen as seperate)
small d= better resolution

d=?/2NA

Diplococci 

divide in one plane

Steptococci

Divide in one plane

Sarcinae 

divide in three planes 

basic, positively charged chromophore

Ex: Methylene Blue and Crystal Violet

acidic, negativley charged chromophore

EX: Acid Fuchsin, Congo Red, Nigrosin

no charge

Ex: Sudan black

Water insoluble

EX: India ink 

Negative stain

size and shape

stains the background, not the cell.

(NOT DARK FEILD MICROSCOPY)

two dyes used:

-Nigrosin  (negative so reppeled by neg charge on bacteria)

-India ink (insuluble so doe snot penetrate cell)

-put the dots of bacteria and dye then spread with another slide at 45 degree angle

DO NOT RINSE OR HEAT FIX

Simple stain 

shape

one dye used to stain all cells the same color

Cationic dyes (methelyne blue and crystal violet)

Gram stain

Differential: based on cell wall

Dye: Crystal violet

Mordant: Iodine

Decolorizer: Ethanol

Counter stain: Safranin

Gram+ Purple

Gram- pink 

Acid fast stain

Differential: based on cell wall (Mycolic wax content)

Primary stain: Carbol Fuchsin 

STEAM

Decolorizer: Acid alcohol

Counterstain: Methylene Blue (cationic)

(High wax) ACID FAST+  RED

ACID FAST- BLUE

Spore stain

structural (specialized)

Primary stain: Malachite Green

STEAM

Decolorizer: water

Counterstain: Safranin

Endospore: green in center of pink 

sporangium: pink

free spore: green oval bodies 

AGAR:

Broth: liquid

Slant: Solid

Deep: solid 

Phenylethyl Alcohol Agar

Selects for growth of gram+

Desoxycholate Agar DES 

selective and differential

selects for growth of gram-

red colony: lactose+

nonred colony: Lactose-

Eosin Methylene Blue (EMB)
SELECTIVE AND DIFFERENTIAL

selects for gram-

Differentiation:

Dark colony: lactose ferm & produce alot of acid 

pink colony: lacrtos ferm

no color absorbed: lact-

Blood agar plate 

differentiates based on reactions to blood

"BAG"

A: Beta hemolysis 

b: Alpha hemolysis

c: Gamma hemolysis

tests for amylase

Indicator: iodine (added after growth)

colorless around colony: +

purple around colony: -

(because iodine+starch=purple so this means th amylase was not there to hydrolize the starch)

tests for caseinase (Hydrolizes casein-Milk)

A: -

B: +

tests for lipase

Blue dye intensifies: + (lower pH)

no blue intensification:-

Phenol Sugar Fermentation and Durham tube

(the first tube shows the control)

yellow: ferm+

lt orange-red:ferm-

Gas in tube: Gas prodctuction

Methyl Red

tests for mixed acid fermentors

indicator: Methly red (after growth)

dye stays red: + pH under 5.1

dye turns yellow: -

VP

test for 2,3 butanediol fermenter

Reagents=

Barrit's reagents.

VP1: Alpha-nahthol

VP2: KOH

red on top:+

no red:-

Catalase

differential

detects catalase

bubbles:+

no bubbles:-

Oxidase

differential: detects oxidase whih removes hydrogen

aromatic ameine used

 (oxidase reagents added after growth)

colorless:-

dark purple/red to dark blue:+

Nitrate broth

Differentail: tests for nitrate reductase

red w/o zinc: +

red w/ zinc: -

Never red: +

Tryptone

differential tests for indole (to see if tryptonase is present because it turns tryptone into indole )

Indicator: Kovac's reagent

red top: indole+ so tryptonase +

yellow top: indole- so tryptonase -

Urea

Differential

detects urease 

indicator: phenol red

Red to cerise=+

yellow to orange=-

SIM

Sulfur Indole Motility 

H₂S positive: Black

Indole+: Kovac's reagent turns red after addition

Motility+: growth away from stab line 

Simmons citrate slant

differential

detects for the use of citrate as sole carbon source 

starts green 

No growth:- (reagardless of color change)

Growth and Blue color: +

differential

 tests for presence of phenylalanase

indicator: ferric chloride added after growth

green: +

no color change to ferric chloride: -

Gelatin

detects gelitinase 

resolidification: -

no resolidification: +

OF GLUCOSE 

Tests to determine if a bacteria can use glucose in an oxidative (aerobic) or fermentative  (anerobic) condition.

On open tube and one closed (added oil)

Results:

-OPEN CLOSED: Incompletly Oxidative (O)

-OPEN CLOSED: Strictly Fermentation (F)

-OPEN CLOSED: Strictly Oxidative

-OPEN CLOSED: Faculative 

KIA
tests for the ability to ferment glucose and/or lactose, tests H2S production and can be used to test for gas production.

 Results:

;Red slant/Red butt: Gluc- Lac- H₂S- 

;Red slant/ yellow butt: Gluc+ Lac- H₂S- 

;Yellow slant/ Yellow butt: Gluc+ Lac+ H₂S- 

;Black Red slant tip and gas: Gluc+ Lac- H₂S+ (Salmonella Proteus) 
; Black yellow slantand gas: Gluc+ Lac+ H₂S+

*NOTE: IF THERE IS GAS AT THE BOTTOM (AND ONLY THE VERY TIP TOP IS RED) THEN IT IS LAC+

Barrit's reagents.

VP1: Alpha-nahthol

VP2: KOH

usually performed in milk. Much quicker than a standard plate count

1.Count the number of organisms seen in a single microscopic field of view

2.Count 9 additional fields of view

3.Add the number of organisms and divide the total by 10 to determine the average

4.Multiple the average by the Microscopic Factor previously determined

# Bacteria/ml = Average # bacteria X MF

Bacteria incubated in a small tube of plasma overnight.

If the plasma becomes clumpy or solidfies, the coagulase+

ONLY VALID ON GRAM+ STAPHYLOCCUS LIKE BACTERIA

• Staphylococcus-
• Aureus (Manitol/caogulase/DNase +)
• Epidermis (Manitol/caogulase/DNase -)
• Streptococcus
• Pneumoniae (Alpha hemolysis)
• Pyorgenes (Beta hemolysis) 

• Corynebacterium
• lactobacillus
• Sporosarcina
• Micrococcus
• Staphylococcus