lab 2 Flashcards
Select the ingredients in nutrient broth
Meat extract
Peptone
Purified Water
Agar
Meat extract
Peptone
Purified Water
During proper aseptic technique when transferring microbes from a broth tube, the rim of a tube is heated to kill microbes only after obtaining the sample from the tube.
TRUE OR FALSE
FALSE
In the Gram staining technique, a Gram positive microbe with a thick peptidoglycan layer will retain what stain components?
Select Answer:
Crystal violet - Iodine complex Crystal violet iodine alcohol safranin
Crystal violet - Iodine complex
TM means Total Magnification. On your Leica microscope used in lab, what is the TM when using the oil immersion objective?
1000x
10x
100x
400x
1000x
The correct abbreviation for Streptococcus Pyogenes is correctly written as S. Pyogenes
TRUE OR FALSE
FALSE
Proper storage of the microscope includes (select all that apply)
- 4x objective in the down position
- Light should be turned off
- Raise the stage all the way in the up position.
- The proper orientation of the microscope in the cabinet includes: orange handle facing inward, oculars face towards the cabinet door
- Oil on the 40x objective should remain on the objective do not remove it.
Light should be turned off
4x objective in the down position
Of the 4 quadrants in a streak plate, will the number of bacterial colonies be more or less in the last quadrant (Q4) streaked following incubation. SELECT THOSE THAT APPLY -MORE -LESS -SAME
Less
The concentration of bacteria in the last quadrant streaked (following incubation) will be less. This is a dilution method and the goal is to have isolated colonies in the last quadrant.
When viewing Gram stained bacteria, what is the Gram reaction of red stained bacterial?
- Gram negative
- Gram positive
- Gram neutral
- Gram variable
Gram negative
In the Gram stain procedure, it is important to transfer a large amount of microbes to the microscope slide.
TRUE OR FALSE
FALSE
The ideal goal in the streak plate technique is to get individual colonies (isolated colonies) in which quadrants?
(select all that apply)
Q1
Q2
Q3
Q4
Q3
Q4
When a slide is clipped in on the stage, there is a glass lens directly under the slide that refracts the light into a cone-shape to increase the amount of light directed toward the specimen. What is the name of this lens?
X and Y traveling knobs Iris diaphragm Condensor high dry objective light source ocular tube
Condensor
When using your Leica microscope, light can be controlled when viewing a slide by adjusting the
(select all that apply)
condenser height knob
Brightness control knob
Ocular diopter
Iris Diaphram
Brightness control knob
Iris Diaphram
Use of Oil and Oil Objective rules
- Always use oil with 100X oil objective
- Only focus with the fine focus knob
- From “home position”, rotate fine focus no more than one full turn in either direction.
- If you lose your sample with oil obj., then go back to 4X and 10X
-but remember, NEVER use or rotate by the 40X objective if oil on slide.
Positive Stain
Positive Stain (use of positively charged stain)
Negative Stain
Negative Stain (use of negatively charge stain)
Simple stains
(one stain used) – all cells are stained the same way -Positive Stain (use of positively charged stain)
- Negative Stain (use of negatively charge stain)
- involves air drying & heat fixing the sample on the slide, this can be used using positive (basic) or negative (anionic) stains.
Gram stain
(use of multiple stains)
cells stain differently; the most important stain technique used in medical field
Structural stains:
1) Capsules stain,
2) Flagella stain
A ______ stain that shows differences in peptidoglycan layer of bacteria.
differential stain
differential stain uses _______ reagents, including _________ stains.
thin peptidoglican stains what color? positive or negative Gram?
thick peptidoglican stains what color? positive or negative Gram?
- Uses 4 reagents including 2 stains.
- Divides the majority of bacteria into two groups:
- Gram positive →thick peptidoglycan →purple stained cells
- Gram negative →thin peptidoglycan → red stained cells
- This is a common first step in the identification of a bacteria, and drug treatment options.
differential stain uses:
_______ reagents, including _________ stains.
Divides the majority of bacteria in 2 groups. what are they?
- Gram positive →thick peptidoglycan →purple stained cells
- Gram negative →thin peptidoglycan → red stained cells
A differential stain that shows differences in peptidoglycan layer of bacteria.
WHAT ARE THE DIFFERENCES?
- Gram positive →thick peptidoglycan →purple stained cells
- Gram negative →thin peptidoglycan → red stained cells
TRUE OR FALSE:
differential stain is a common first step in the identification of a bacteria, and drug treatment options.
TRUE
Gram-positive bacteria have a THICK peptidoglycan layer
TRUE OR FALSE
TRUE
Gram-positive bacteria have a THIN peptidoglycan layer
TRUE OR FALSE
FALSE
Gram-negative bacteria have a THIN peptidoglycan layer
TRUE OR FALSE
TRUE
Gram-negative bacteria have a THICK peptidoglycan layer
TRUE OR FALSE
FALSE
Crystal violet
PURPOSE?
GRAM NEGATIVE COLOR? GRAM POSITIVE COLOR?
-Primary Stain – stains all cells purple GRAM NEGATIVE COLOR: purple GRAM POSITIVE COLOR: purple
iodine
PURPOSE?
GRAM NEGATIVE COLOR? GRAM POSITIVE COLOR?
-Mordant – binds to crystal violet, forms a CV-I complex GRAM NEGATIVE COLOR: purple-blue GRAM POSITIVE COLOR: purple-blue
95% Alcohol
PURPOSE?
GRAM NEGATIVE COLOR? GRAM POSITIVE COLOR?
-Decolorizer – rinses complex out if organism has a thin peptidoglycan layer GRAM NEGATIVE COLOR: colorless GRAM POSITIVE COLOR: purple-blue
Safranin
PURPOSE?
GRAM NEGATIVE COLOR? GRAM POSITIVE COLOR?
-Counterstain – stains any unstained cells pink-red GRAM NEGATIVE COLOR: pink-red (result) GRAM POSITIVE COLOR: purple-blue (result)
in what order should we use the following Reagents? IODINE CRYSTAL VIOLET SAFRANIN 95% ALCOHOL
CV
IODINE
95% ALCOHOL
SAFRANIN
PROCEDURE FOR Observing CELLS ON MICROSCOPE
- Place slide on stage
- Move stage so sample is centered
- 4x – focus/center on predominant stained zone (Red or Blue) →But also pick an area of WEST MICHIGAN, not NEW YORK CITY
- 10x – add light →focus →re-center
- 40x – add light →focus →re-center
- 100x oil – add light →focus →re-center
what are the most common arrangement patterns of morphology?
diplococci streptococci staphylococci bacillus streptobacilli
how many quadrants do we use on a streak plate technique?
all 4
what is the purpose for Streak Plate Isolation Technique?
- Determine if any contaminants in broth culture.
- Ensure continued use of pure culture.
- Observe colony morphology
what is the goal for Streak Plate Isolation Technique?
Isolated colonies in 3rd and 4th quadrants.
Methods Used to Determine Motility
- Flagella stain: non-viable cells are stained to highlight flagella. (DIRECT EVIDENCE OF MOTILITY)
ref: Atlas pg54 - Use of an agar deep (Example: SIM medium): semi-solid medium where motile bacteria can move (indirect evidence of motility)producing a turbid (cloudy) area in the medium.
- Hanging drop – live bacteria move freely in a liquid medium (indirect evidence of motility)
SIM Agar Tube:
What does S stand for?
What does I stand for?
What does M stand for?
S: sulfide
I: indole
M: motility
SIM Agar Tube:
Growth away from stab line indicates organism is ______.
motile
Observation of Bacterial Capsule using Specialty Capsule stain:
uses what 2 stains?
which is positive?
which is negative
- Positive charged safranin →stains the cell (not repelled by the cell, but penetrates it - thus stain the net negatively charged cytoplasm of the cells)
- Negative charged (nigrosin) → stains the glass background outside of the capsule or cell. It is repelled by the negatively charged surface of the bacteria.
(ex: Klebsiella sp.)
ref: Atlas pg51
Observation of Bacterial Capsule using Specialty Capsule stain:
A _______around the cells is evidence of the capsule.
What is the relevance?
clear zone
relevance: capsules cloak the surface features that can be recognized by the immune system.
Methods Used to Determine Motility in Motility SIM Agar tubes.
How does non-motile bacteria appear?
stay near the stab (flat growth)
Methods Used to Determine Motility in Motility SIM Agar tubes.
How does motile bacteria appear?
motile bacteria move throughout the medium (360°) - and cause the medium to become cloudy
Is the SIM agar tube only used for motility?
NO
What is the SIM agar tube used for?
- motility
- sulfide
- indole
capsule stain:
is nigrosin negative or positive stain?
what is it’s purpose?
appearance?
negative
- capsule stain repelled by the negatively charged surface of bacteria
- appearance: clear area around the cell is the capsule
- (also stains background)
capsule stain:
is safranin negative or positive stain?
what is it’s purpose?
appearance?
positive
- penetrate the cell
- stains net negatively charged cystoplasm of the cells
What does RODAC stand for?
Replicate Organism Detection And Counting
what does CFU stand for?
colony forming unit
each colony was formed by CFU’s which is divided many times (binary fission)
what is the purpose of using a RODAC plate?
to calculate CFU’s
number &; diversity of colonies
what do you include when recording Gram reaction statement?
- Gram reaction: negative or positive
- Morphology/Arrangement (if present) (ex: coccus, staphylococcus, et)
- Presence of endospores (if present) (recorded as “with endospores” or “with spores”)
- Presence of body cells (if present) ex: WBC’s, RBC’s, neutrophils, other
- note if microbes are “intracellular” in the host cells (ex: intracellular Gram negative diplococci in neutrophils, etc)
Photosynthetic microbes include only eukaryotes.
TRUE OR FALSE
FALSE
What are the 3 SIM tests used for?
S= sulfur production I= indole production M= motility
What three lab procedures are used to determine if an organism is motile?
Hanging drop, flagella stain, and SIM medium.
Name a basic (cationic) dye
Methylene blue, crystal violet, safranin
Name an anionic dye.
Nigrosin
Name a genus that is a Gram positive bacillus.
Bacillus sp.
Name a genus that is a Gram negative bacillus.
Escherichia sp.
Name two sterile body sites.
Uterus, inner ear…
What type of microscopy is best used to highlight the morphology of spirochetes?
Darkfield microscopy
Which staining technique presents cells in a more natural state?
Negative staining technique. The cells are not heat or chemically modified.
Name a medium that is selective and differential. Explain the role of the chemicals found in this medium with reference to its selective and differential abilities.
MacConkey agar contains both growth inhibitors and stimulators. It is selective for Gram negative non-fastidious bacilli. It differentiates between organisms that are lactose fermenters and non-lactose fermenters. Fermenters catabolize the lactose producing acids. The acids decrease the pH of the medium and cause the pH dye to change color producing a dark pink to red color change.
MacConkey agar selects for ____ ____ species.
Gram negative
List the size order of the following cells: erythrocyte, yeast cell, Staphylococcus sp., neutrophil.
Neutrophil; Erythrocyte; Yeast cell; and Staphylococcus sp.
Names and Classification of Media
General
general purpose media supports growth of many non-fastidious organisms.
Names and Classification of Media
General purpose media types
Nutrient agar, Nutrient broth
Names and Classification of Media
Enriched
like general media, but enriched with specific nutrients to promote the growth of fastidious organisms.
Names and Classification of Media
name an enriched purpose media type
Blood agar
Names and Classification of Media
Selective
grows specific bacteria, contains inhibitor(s) preventing growth of others.
Names and Classification of Media
name Selective media types
MacConkey agar, Sabouraud dextrose agar
Names and Classification of Media
Differential
Differential - shows differences between organisms that grow on it.
Names and Classification of Media
name differential media types
Blood agar (know, but to be explained after exam 1) , MacConkey agar
Nutrient agar and broth (NA, NB) is classified as what type media?
General - general purpose media supports growth of many non-fastidious organisms.
MacConkey agar (MAC) is classified as what type media?
Selective - grows specific bacteria, contains inhibitor(s) preventing growth of others.
& also
Differential - shows differences between organisms that grow on it.
Sabouraud dextrose agar (SDA) is classified as what type media?
Selective - grows specific bacteria, contains inhibitor(s) preventing growth of others.
Blood Agar (BA or SBA) is classified as what type media?
Enriched – like general media, but enriched with specific nutrients to promote the growth of fastidious organisms.
& also
Differential - shows differences between organisms that grow on it. (know, but to be explained after exam 1)
name the 7 Stain / reagents List
Nigrosin Methylene blue Crystal violet Safranin Lactophenol cotton blue Gram iodine 95% alcohol
name the 5 Biochemical Tests
Glucose and Lactose (carbohydrates) broth SIM (Sulfide, Indole, Motility) Citrate slant Urea broth MacConkey Agar – Lactose fermentation
what is Bibulous paper used for?
For drying slides with no cover slip
For removing oil on slides with no coverslip
what is Lens Paper used for?
use on all oculars/objectives / if paper gets oily, use different lens paper so oil is not transferred to non-oil areas.
Lens cleaner solution used for?
okay to use on all oculars/objectives / dab on lens paper or swab.
Kimwipes used for?
use on all but never oculars/objectives.
Use of Oil and Oil Objective
Always use oil with 100X oil objective
Only focus with the fine focus knob
From “home position”, rotate fine focus no more than one full turn in either direction.
If you lose your sample with oil obj., then go back to 4X and 10X
but remember, NEVER use or rotate by the 40X objective if oil on slide.
At end of EVERY lab, follow SOP for clean and storing your microscope / check 40X obj for oil.
basic (positive) stains name 3 charge? what do they do? drying technique?
methylene blue, crystal violet, safranin
+ charge molecules which bind to negatively (-) charged cell envelope and cytoplasmic structures of microbial cells. The basic simple stain involves air drying and heat fixing
-in some specialty applications, such as with capsule stain, both positive and negative stains are used together (to show bacterial capsules)
Negative stains
name 1
what do they do?
drying technique?
- nigrosin
- negative charged molecules that are repelled by the negative charge of the cell envelopes of microbes. as such, the stain only colors the background around the cells. this is often employed with cells known to be very fragile and basic staining may damage them.
- after staining, the cells look like clear spots on a stained background.
- does not involve air drying or heat fixing
- in some specialty applications, such as with capsule stain, both positive and negative stains are used together (to show bacterial capsules)
who developed the Gram stain?
Christian Gram
Each colony is a result of the growth from one ____
bacterium
how long do you sterilize the inoculating loop for? how long cool?
15 seconds each
room temperature
approx 25C
body temp
approx 37C
some, not all bacteria have structures that enhance their ability to cause disease. Name 3 and explain why they may make the microbe a better pathogen
- pili - attachment pili- attach organism to tissue
conjunction pili - transfer genetic material (plasmid genes)- transferring drug resistance to microbe ( enhance the ability to remain) - flagellum - move organism & escape immune defenses
3.. glycocalyx (capsule & slime layer) used as cloaking device so blood cells don’t engulf it - primarily due to net negative charge of cells - the two repel each other
glycocalyx - form biofilm with other microbes which increases the difficulty level of treatment protocols - plasmids- contribute to overall survival of organism - protein synthesis of plasmid genes that involve drug resistance, toxin production.
you have just obtained a wound swab from a patient. the dr wants to know within 2 hours if the specimen is mixed or pure. what procedure would you use and how could you tell if the specimen where pure or not?
Gram stain technique.
if specimen was pure - microbes would be purple or pin, not bother & would show bacteria of all one morphology
what is Brownian movement when looking for motility?
movement occurs because water molecules bombard the microbe & result in slight movement of the microbe - not evidence of motility.
