Weeks 5 Differential Stains and Effect of Temperature

What type of stain is a gram stain?

Differential

Differentiates Gram Negative and Gram positive organisms

What has a thicker peptidoglycan layer, gram negative or gram positive bacteria?

Gram positive

Which bacteria type has two membranes?

Gram Negative

When stained what color does gram postive bacteria give off?

Purple

When stained what color does gram negative bacteria give off?

Pink

What is the second layer of cell wall made of that gram negative bacteria have 

LPS (Lippopolysaccharide)

Since gram negative Bacteria have two outer cell walls what are they resistant to?

Large molecules such as protein, so they are more resistant to large antimicrobial drugs 

What are the steps for gram stain?

1)MAke a smear then, Stain culture with primary stain crystal violet

2) Wash Crystal Violet Off with deionized water\

3) Use mordant, Iodione on slide

4) Wash off Iodine

5) Add Alchohol (decolorizer) Only for a bit 

6) Add Counterstain Safranin 

In the gram stain what does the primary stain crystal violet do 

Basic dye stians positive 

Stains all bacteria purple 

IN the gram stain what does mordant iodione do?

Reacts with crystal violet to form complex that make it harder for crystal violet to be removed from peptidoglycan layer

The thicker the peptidoglycan layer, the better it can retain crystal violet 

In the gram stain what does the alcohol, decoloizer do?

Removes Crystal violet complex

Gram Negative complex falls out quicker due to it’s thinless allowing it to have a pink color 

In the gram stain what does the counterstain saffrinin do?

Since both types of bactera lost their crytsal violet complex (gram negative more)

They can take saffrinin in their peptidoglycan, however since postive alr has crystal violet, it wont take as much

Why can gram stain go wrong?

1) Some bacteria lack a cell wall that can even gram onto dyes

2) Acid fast bacteria, can’t cling onto dye well

3) Old culture

What is the KOH test (String Test) 

Used to further detect if organism is gram postive or gram negative

Gram Negative bacteria thin cell die when placed in KOH solution causing cell to lysis

DNA cums out, it looks like cum its sticky and long

What is a positive KOH test

Gram Negative Bacteria only will give a postiive KOH test

What is a negative KOH test?

Gram positive bactiera have strong peptidoglycan cell wall so they won;t cum

When doing a KOH test where are you supposed to get your colonies from?

A solid colony, not from broth as it is not enough

What temperature does Psychrophiles grow at the best?

-10°C to ~20°C

Artic and fridge

What temperature do Mesophiles grow at the best?

10–50°C

Human pathogens

What temperature do Thermophiles grow at the best?

40–70°C

Compost, hot springs

What temperature do Hyperthermophiles grow at the best 

65–110°C

What is the optium temperature for microorganisms?

Where Growth Rates are the highest 

What is the minimum growth temperature?

The lowest temperature where microbes can reproduce at and live and shit

What is the maximum growth temperature?

Highest temperature where microbes can reproduce at

What is thermal death time?

How long a organism can live at a certain time

What is Thermal death point?

Minimum temperature it takes to kill microbe in a specific amount of time

What is pasturization

Heat required to kill organisms that cause early spoilige, but not kill the product 

What happens when organisms get into an environment below their optimum temp?

molecule movement and enzyme function slows  

reduces the fluidity of the membranes

What happens to organiams that experince a enviroment above their optimal temp?

Denature

Loss of cell integrity 

What type of test is a acid fast stain

Differential

Used to detect Mycobacteria

What is special about mycobacteria, and why do we need to use an acid fast stain in order to detect them?

Gram positive

Mycolic acid surrounds their wall, this makes them waxy

They retain dyes really well

What are the steps for an Ziehl Neelsen acid fast stain?

1) Create smear with positive mycobacteria, negative mycobacteria and mix

3) Places onto heating trail with foil, cover with towel, stain with primary stain Carbolfuchsin

4) Remove slide and bring back to table rinse off stain 

5) Decolorize

6) Add counterstain mehylaine blue

Expalin what was the primary stain in the acid fast stain, and what does it act on?

Carbolfuchsin, it works by binding really well to the walls of acid fast bacteria and can’t easily be removed

What is the counterstain in the acid fast shit?

Methylaine blue, it stains blue

What is a endospore stain?

Differentiates endospores from vegetative cells

What are the steps for a endospore stain?

1) Create a smear

2) Bring smear to fume hood, cover slide with towel

3) Use primary dye Malachite Green

4) Rinse off and decolorize it

5) Use counter stain safrinin

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What do endospores look like

Green

What do vegetative cells loook like

pink

Why do you need heat for a endospore stain?

Heat helps primary stain malachite green penetrate… through the thick spore coat and cell.