Unit 5 :staining techniques

Question 1

what are the 2 main differential groups in principle of Gram stain

Answer 1

Gram-positive and Gram-negative

Question 2

what is the difference between Gram-positive and Gram-negative organisms in terms of cell wall

Answer 2

Gram-positive =thicker cell wall with more peptidoglycan and

Gram-negative = additional outter membrane instead of thick cell wall,

Question 3

what separates the cell wall and the outer membrane of the Gram-negative organisms

Answer 3

periplasmic space

Question 4

which species of microorganisms do Gram stain stain for

Answer 4

bacteria

Question 5

list the 4 steps involved in Gram staining

Answer 5

1.primary stain (crystal violet)
2.trapping agent (Gram’s Iodine)
3.rapid decolourisation (alcohol/acetone)
4.counterstaining (safranin)

Question 6

explain what crystal violet does to both Gram-positive and Gram-negative cells and the colour change

Answer 6

-crystal violet penetrates through cell walls & cell membranes of both Gram-positive and Gram-negative cells

-stains cells purple

Question 7

what does Iodine do

Answer 7

reacts with crystal violet to form large crystal-iodine complexes

Question 8

what does alcohol/acetone do

Answer 8

alcohol reacts with the lipids of the cell membrane

Question 9

what are the different reactions from Gram-positive and Gram-negative cells to decolorization by alcohol

Answer 9

Gram-negative cells lose their outer membrane along with the crystal-iodine large complexes
(they are washed away)

Gram-positive cells become dehydrated and the large iodine-crystal violet large complexes become trapped within the multi-layered peptidoglycan cell wall

Question 10

what is the colour status of Gram-negative and Gram-positive cells after decolorization by alcohol

Answer 10

Gram-positive - retain purple colour

Gram-negative -lose their purple colour

Question 11

after counterstaining by safranin, what is the colour status

Answer 11

Gram-negative - pink-red colour

Gram-positive -purple colour

Question 12

what are the results expected for Gram stain as per principle

Answer 12

Gram-positive = purple

Gram-negative = reddish pink

Question 13

Now give the Gram’s stain principle, process, reaction and expected results off by heart

Answer 13

Principle : it differentiates bacteria into Gram-positive (thicker cell wall) and Gram-negative (thin cell wall with an additional outer membrane) based on their cell wall.

Process :
-Primary stain by Crystal violet stains both Gram-positive and Gram-negative cells purple.
-Trapping agent, Gram’s Iodine reacts with Crystal Iodine to form large crystal-Iodine complexes in both Gram-positive and Gram-negative cells
-Decolorization by alcohol/acetone washes away the additional outer membrane + large complexes in Gram-negative cells and dehydrates the Gram-positive cells and results in large complexes being trapped in the multi-layered peptidoglycan cell wall. Gram- negative cells lose their purple colour and Gram-positive cells retain their purple colour.
-Counterstaining by Safranin gives the Gram-negative a reddish pink colour while Gram-positive cells remain puple.

Expected results :
Gram-positive =purple
Gram-negative =reddish pink

Question 14

give 4 examples of gram-positive organisms.

Answer 14

Streptococcus
Staphylococcus
Bacillus
Clostridium

Question 15

2 examples of gram-negative organisms

Answer 15

Proteus
Neiserria

Question 16

what is the principle for the ziehl-Neelson staining technique

Answer 16

it differentiates acid-fast organisms and non-acid-fast bacilllus

Question 17

what is meant by Acid-fastness

Answer 17

physical property of bacteria to resist decolorisation by acids during staining procedures.

Question 18

what does a decolorizer do

Answer 18

it dehydrates and rinses out

Question 19

what are the 3 reagents used in Ziehl-neelson staining technique

Answer 19

Ziehl-Neelson carbolfuchsin
acid alcohol
methylene blue

Question 20

what 2 pre-steps are required before attempting to penetrate high lipid cell walled mycobacteria such as bacillus

Answer 20

phenol (which is lipid soluble)
heat application (melts the wax)

Question 21

what step follows addition of phenol and heat application to bacillus

Answer 21

primary staining by Ziehl-Neelson Carbolfuchsin

Question 22

what step, and what is acid alcohol used for

Answer 22

step3 for decolorization

Question 23

how does bacillus react to decolorisation by acid alcohol

Answer 23

Acid-fast bacillus withstands and it retains the Ziehl-Neelson carbolfuchsin stain and non-acid-fast bacillus doesnt.

Question 24

what is the last step and what is the expected colour change

Answer 24

counterstaining with malachite green or methylene blue.

expected colour change:
acid-fast bacilli - red
non-acid- fast organisms & cells - green or blue

Question 25

give 1 example of acid-fast bacillus

Answer 25

mycobacterium tuberculosis

Question 26

state the principle, 3steps and expected colour change involved in Ziehl-Neelson staining technique

Answer 26

principle: it differentiates bacilli into acid-fast and non-acid fast organisms. 1. heat application (to melt wax) and phenol application (for lipid-dense cell wall) 2. primary staining with Ziehl-Neelson carbolfuchsin and both acid-fast and non-acid fast stain red. 3. decolorization with acid alcohol results in acid-fast bacilli retaining their red colour and non-acid fast organisms and cells being decolorized. 4. counterstaining with malachite green or methylene blue Expected colour change acid-fast bacilli: red background, non-acid fast organisms or cells: green or blue

Question 27

what 2 methods are used to stain endospores

Answer 27

ziehl-neelson stain and malachite green stain for spores

Question 28

why do we say endospores tend to resemble acid-fast behaviour

Answer 28

when bacilli is stained with ordinary dye or with Gram stain it stains deeply but its endospores resist ( due to their thick resistant spore coat) the dye and appear as a clear area in the organism. and when stained endospores tend to retain the dye even after decolorization thus acting like an Acid-fast organism

Question 29

what makes the Ziehl-neelson staining be able to stain endospores while ordinary stains cant, what is it that it has that enables this, what makes it special

Answer 29

the ziehl-neelson primary stain (ziehl-neelson carbolfuchsin) contains phenol which softens the protein coat and the application of heat helps drive the stain into the endospore

Question 30

what is used as a decolorizer in the Ziehl-neelson staining of endospores

Answer 30

2% nitric acid in absolute ethanol

Question 31

what are the expected results from the malachite green and the ziehl-neelson staining of endospores

Answer 31

ziehl-neelson stain: spores-bright red cytoplam or rest of the cell-blue

Question 33

what are the 3 steps involved in malachite green staining of endospores

Answer 33

1.primary stain is malachite green which stains the entires cell and spores green 2.heat is used to force the stain into the spores 3. primary stain washed away from cell wall with water but retained in the endospore wall 4.counterstain staining with safranin to stain the vegetative cell/cytoplasm

Question 34

does the dye bind the same way on the vegetative cell as it binds to the endospore wall

Answer 34

no. it binds relatively weakly to the vegetative cell wall and is removed when washed with water while it is locked in in the spore.

Question 35

what are the malachite expected results

Answer 35

spore: green vegetative cells/cytoplasm : red and lipid granules are unstained

Question 36

describe the shape of the following types of bacteria 1. cocci 2.bacilli 3.vibrio 4.spirillum 5.spirochete

Answer 36

1. cocci- spherical/oval 2.bacilli-straight rod shaped 3.vibrio - curved rod shaped 4/5. spirilla- spirally twisted

Question 37

how would you identify the following types of cocci 1.diplococci 2.strepococci 3.staphylococci 4.micrococci 5. sarcina

Answer 37

1.diplococci- paired cocci (spherical) 2.strepococci -chains 3.staphylococci-irregular clusters 4.micrococci- groups of 4 cocci 5. sarcina- cubical packets of 8 cocci

Question 38

how would you describe the following types of Bacilli 1.coccobacilli 2.bacilli 3.palisades 4.diplobacilli 5.streptobacilli

Answer 38

1.coccobacilli- short rods approaching coccus shape 2.bacilli- 1 bacillus cell 3.palisades-group of vertical bacilli 4.diplobacilli-2 bacilli connected together 5.streptobacilli- chain of bacilli

Question 39

difference between spirochaetes and spirilla bacilli

Answer 39

spirochaetes are flexious and are motile without a flagella

Question 40

are yeasts unicellular or multicellular and how would you describe their shape

Answer 40

unicellular fungi oval or spherical single cells

Question 41

how do yeats reproduce

Answer 41

by asexual reproduction called budding producing blastospores

Question 42

are yeasts Gram+ or Gram-

Answer 42

gram+

Question 43

draw a unicellular yeast, budding yeast, unicellar yeast next to a blastospore

Answer 43

answer on workbook

Question 44

how would you define/describe yeast-like fungi

Answer 44

they grow partly as yeasts partly as long filamentous cells joined end to end, forming pseudo-mycelium

Question 45

what are thick-walled resting spores of yeast-like fungi called

Answer 45

chlamydospores

Question 46

give an example of yeast-like fungi

Answer 46

candida albicans

Question 47

what is hypha

Answer 47

the connection between 2 yeast-like fungi as they are joined together end to end

Question 48

how would you describe true-fungi

Answer 48

1.they grow as long filaments or hyphae which branch and interface to form mycelium. 2.they reproduce by spores 3.vegetative mycelium- penetrates into substrate, absorbing nutrients for growth 4.aerial mycelium protrude from the vegetative mycelium into the air, form and release spores

Question 49

how does dimorphic fungi grow at 37degrees and at 22degrees and which of the 2 temperatures is favourable and which one is unfavourable

Answer 49

37degrees in the body (favourable) -yeasts (oval or spherical) 22degrees in artificial media (unfavourable)- moulds (long filaments)

Question 50

go to notes (unit 5, last page) and label the different types of spores

Answer 50

answer on notes

Question 51

when preparing a dry smear, you need to leave the sample on the glass slide to dry for a few minutes before continuing with the experiment, explain why you cant speed up this process by blowing or heating

Answer 51

by blowing we will be forcing the bacteria into the air and this will contaminate and cause a possible infection

Question 52

explain heat-fixing

Answer 52

heat fixing is passing a tilted and dried slide 2-3 times over a heat to make the bacteria to stick to the slide.

Question 53

what do we use to stain mycobacterium tuberculosis

Answer 53

ziehl-neelson stain

Question 54

define an acid-fast organism

Answer 54

an organism that has a waxy layer covering their cell wall and thus resists decolourization by acids

Question 55

which staining technique is used for acid-fast bacteria (the ones that resist decolourization by alcohol or acids)

Answer 55

ziehl-neelson technique

Question 56

whats that thing thats used to provide a flame in heat fixing process and what is used to pick a smear from the broth or agar

Answer 56

bunsen burner wire loop

Question 57

what colour is the acid fast bacteria

Answer 57

background : blue and mycobacterium tuberculosis : red/purple bacilli

Question 58

(ngwaneso just cram) what colour does phenol red change to when bacteria has fermented an acidic glucose

Answer 58

yellow

Question 59

if bacteria didnt ferment carbohydrate what is the colour change and how do we know if gas is produced

Answer 59

it remains red the tube will have a bubble

Question 60

which bacterial organism is a good example of bacteria that has endospores and which stain is used to stain the endospores

Answer 60

bacterium cereus ziehl-neelson stain and malachite green

Question 61

what is the staining reaction and the organism shape

Answer 61

Ziehl-neelson stain: blue vegetative organism and red endospores Malachite green stain: red vegetative organism and green endospores ... ...which are bacilli in shape