Lab Practical Part 1 Flashcards
Bacterial species have characteristic patterns of colony growth, but can this be used as a diagnostic tool to identify a colony on a plate or broth?
- Morphology gives clues, but can’t identify the species . Different species night have similarities
- Medium, temp, environment also affect growth patterns
Pinpoint, small, moderate or large
Size
Color of colony
Pigmentation
Shape of colony
Form
Unbroken, peripheral edge
Circular
Indented peripheral edge
Irregular
Cultural characteristics
differences in macroscopic appearance of growth
What is used to separate microorganisms into taxonomic groups?
Cultural characteristics
Rootlike, spreading growth
Rhizoid
The appearance of the outer edge of the colony is called
Margin
Sharply defined, even
Entire
Marked indentations
Lobate
Wavy indentations
Undulate
Toothlike appearance
Serrate
Threadlike, spreading edge
Filamentous
Degree to which the colony growth is raised on the agar surface
Elevation
Elevation not discernable
Flat
Slightly elevated
Raised
Dome shaped elevation
Convex
Raised, with elevated convex central region
Umbonate
A culture containing a single unadulterated species of cells is called a
Pure culture
Cluster of cells that originates from the multiplication of a single cell and represents the growth of a single species of microorganism
Colony
A solution containing these nutrients is a
Culture medium
All culture media are
- Broth culture
- Semisolid medium
Broth medium
Lacks a solidifying agent
Semisolid medium
A concentration of less than 1% agar
A solid can exist as 3 forms as agar
- Agar slant
- Agar deep
- Agar plate
Sterility is the hallmark of successful work in the microbiology laboratory and _______ is the process of rendering a medium or material free of all forms of life
Sterilization
Using correct aseptic technique minimizes the likelihood that
Bacterial cultures will be contaminated
Using correct aseptic technique minimizes the likelihood that bacterial cultures will be contaminated and reduces the opportunity that
you will be exposed to potential pathogens.
Heat = __________or __________such as the autoclave used in class which is adding steam with pressure over time.
Dry (hot air)
Moist (wet heat)
Types of sterilization techniques
- Heat
- Filtration
- Chemicals
- Radiation
normal flora that does not cause negative effects in healthy individuals but instead symbiotic relationships with your skin.
Resident flora
can be removed with good handwashing techniques, even adding surgical scrubbing.
Transient flora
Why are resident flora more difficult to remove?
They are more difficult to remove because they are accustomed to living inside or on the human body. They have evolved and adapted to withstand the conditions of the human body. The immune system might not react to them because it recognizes them as part of the body. They are also covered by hair follicles, oil, and dead skin cells which obstruct their removal.
Clinical Application: What are nosocomial infections?
Nosocomial infections are infections that someone contracts while in a medical facility. It is important to clean surfaces thoroughly and use appropriate protective gear while working in a medical environment so nosocomial infections can be prevented.
How is handwashing affected by water only, soap and water, soap and scrubbing?
- water removes polar
- soap is surfactant, amphipathic
S. scrubbing, physically removes the substances
How does resident flora influence infections?
- Using up nutrients
- outcompete for space and resources.
- Can cause infection when it goes somewhere else
How does handwashing affect residential versus transient flora?
Both types of flora can be removed through handwashing, however, transient flora is more easily removed from the hands than resident flora. Some resident flora resides on the skin and has adapted to be hard to remove from the skin. Resident flora are covered by hair follicles, oil, and dead skin cells which obstruct their removal
If medical and surgical personnel wear gloves, then why do they still have to wash their hands?
There may be an accidental transfer of microbes when removing PPE. Or PPE may not be donned appropriately, exposing the medical personnel and patients to microbes.
subculturing
The transfer of microorganisms from one medium to another.
What are you preparing and maintaining?
We are practicing subculturing to learn to prepare and maintain stock cultures in various media.
What is an inoculum?
An inoculum is a microbial growth that is used to start another culture of that same microbe. This allows microbiologists to examine the growth of the microbe in a specific medium under controlled conditions.
What is inoculation?
inoculation is the process of introducing a microbe to a medium.
Identify three rules that you would tell another student to always remember to do when you are completing an aseptic technique for handling and transferring microbes.
Flame the loop or needle before and after inoculation.
Flame test tubes before and after using inoculating loop.
Never place the caps on the laboratory bench. The caps must be kept in the hand that holds the sterile inoculating loop or needle.
Flaming the inoculating instrument prior to and after each inoculation
These precautions help prevent contamination of the inoculum and help prevent accidental transfer of microbes on other surfaces. They also help protect the microbiologist against contamination.
Holding the test tube caps in the hand as illustrated in Figure 2.1 on page 14
This prevents the spread on microbes onto the lab bench which can lead to accidental infections.
c. Cooling the inoculating instrument prior to obtaining the inoculum
Heat can kill microbes. It is important to cool the instrument to avoid damaging the cultures in the inoculum.
Flaming the neck of the tubes immediately after uncapping and before recapping
These precautions help prevent contamination of the inoculum and help prevent accidental transfer of microbes on other surfaces. They also help protect the microbiologist against contamination.
Identify a pure culture
Pure cultures contain only one type of organism and allow us to study their cultural, morphological, and biochemical properties.
Identify a streak plate
The streak plate technique is a technique used to isolate pure cultures. This dilution technique spreads a loopful of culture over the surface of an agar plate as a means to separate and dilute the microbes and ensure individual colony growth.
In the “Tips for Success” section on page 22, what is the point of number 1?
An isolation plate has isolated distinct, individual colonies. The point of this is to get
individual, isolated colonies somewhere on the plate.
In the “Tips for Success” section on page 22, what is the point of number 2?
2.Pay attention to how well you sterilize your loop and maintain your aseptic technique
If the loop is not sterilized between streaks, or if aseptic technique is not maintained, there will not be a decrease in bacteria leading to individual colonies.
How does this technique apply to Koch’s Postulates? Chapter 1 and Chapter 14 will be helpful to answer this question.
- Isolate, grow and compare the microorganism in people with the same disease.
- Reintroduce in healthy person and see if they get disease
- Singles our microorganism as cause of a disease
The streak plate technique helps make the discrete, isolated, identical bacterial colonies needed to identify bacterial pathogens. which lets observers
It allows observers to collect samples of one type of culture and grow it to observe its characteristics.
____ ______ staining is the use of a single stain, for visualization of morphological shape (cocci, bacilli, and spirilli) and arrangement (chains, clusters, pairs, and tetrads).
Simple staining
Simple staining staining is the use of a ________ stain, for visualization of morphological shape
single
Simple staining staining is the use of a single stain, for visualization of morphological _______ (cocci, bacilli, and spirilli)
shape
Simple staining staining is the use of a single stain, for visualization of morphological shape (cocci, bacilli, and spirilli) and _____________ (chains, clusters, pairs, and tetrads).
arrangement
__________ ___________staining is the use of two contrasting stains, for the separation into groups acid-fast stain and Gram stain, and the visualization of structures (flagella, capsule, spore, and nuclear).
Differential staining
Differential staining staining is the use of _____ contrasting stains, for the separation into groups acid-fast stain and Gram stain, and the visualization of structures (flagella, capsule, spore, and nuclear).
two
Differential staining staining is the use of two contrasting stains, for the separation into groups acid-fast stain and ______ stain, and the visualization of structures (flagella, capsule, spore, and nuclear).
Gram
Differential staining staining is the use of two contrasting stains, for the separation into groups acid-fast stain and Gram stain, and the visualization of ________ (flagella, capsule, spore, and nuclear).
structures
T or F Bacterial smears must be prepared prior to executing of any of the staining techniques.
True
Whether using a broth culture or a culture from a solid medium, both should be air dried before continuing. T o F
True
T o F A broth culture requires water to be added to dilute the broth so it can be spread out to about the size of a nickel.
False
There is no way to avoid the bacterial smear from washing away on the slide. T o F
False
Simple staining methods are a quick procedure for determining
whether a clinical sample has the presence of a foreign bacterial pathogen.
Identify the bacteria that is a chain of spheres
streptococcus
Identify the bacteria that is a curved rod
vibrio
Identify the bacteria that is a chain of rods
streptobacillus
Identify the bacteria that is a cluster of spheres
staphylococcus
Slides should be handled with care during negative staining because
Heat fixation is not applied, leaving the bacteria alive
the first diagnostic test
gram staining
Shape, orientation, gram type, and can be performed on multiple cultures.
gram staining
to determine if an organism possesses a capsule, or demonstrate spore formation
negative staining
frequently used to ID a specific fungi that is found in bird droppings and linked to meningeal and lung infections in humans
negative staining
Gram stains are
differential stains
Imparts color to all cells
crystal violet
The mordant; Intensifies color of the primary stain
Gram’s Iodine
Decolorizing agent; Removes the primary stain
95% ethyl alcohol
The counterstain; Contrasting color to the primary
Safranin
Steps of Gram stain
- Crystal Violet
- Gram’s Iodine
- 95% Ethyl alcohol
- Safranin
Besides the order of staining and the age of the cultures used, name 2 other important factors when completing a gram stain.
1.It is important not to over-decolorize or under-decolorize the smear with the decolorizing agent (ethyl alcohol 95%). Decolorizing too much will make gram-positive cells appear gram-negative. The opposite can occur if the cells are under-decolorized.
2.Slides need to be washed with water between applications of reagents to remove the excess leftover reagent from the previous step.
Thin layer of peptidoglycan is stained
Gram negative
Thick layer of peptidoglycan is stained
Gram positive
Purple in color
Gram positive
pink in color
Gram negative
In an acid-fast stain, only non-acid-fast cells undergo decolorization and look clear, ready to pick up the counterstain to take on its ____color, while acid-fast cells retain the red from the primary stain.
blue
In an acid-fast stain, only non-acid-fast cells undergo decolorization and look clear, ready to pick up the counterstain to take on its blue color, while acid-fast cells retain the ____ from the primary stain.
red
The acid-fast stain is of diagnostic value in identifying organisms that cause _________, leprosy, and other infections.
tuberculosis
The characteristic difference between mycobacteria and other microbes is the presence of
a thick, waxy (lipoidal) wall that makes penetration by stains extremely difficult.
_____ in the form of steam can be used to drive the carbol fuschin into the waxy lipoidal cell wall.
heat
The cells walls of Mycobacteria and Nocardia contain _______ _____and are resistant to penetration by water-soluble stains.
mycolic acid
Members of Clostridium and Bacillus can exist as
1.vegetative cells
2.spores
Members of Clostridium and Bacillus genera are examples of organisms that have the capacity to exist either as metabolically active ________ cells or as highly resistant metabolically inactive cell types called spores ready for more favorable environmental conditions.
vegetative
are spores metabolically active?
No, spores are metabolically inactive cells
Some spore forming bacteria can have extremely negative health effects. These bacteria include ________ anthracis, which causes anthrax, and certain __________ bacteria which are the causative agents of tetanus, gas gangrene, food poisoning, and pseudomembranous colitis.
Bacillus
Clostridia
Some spore forming bacteria can have extremely negative health effects.
Bacillus and clostridia
causes anthrax,
Bacillus anthracis,
causative agents of tetanus, gas gangrene, food poisoning, and pseudomembranous colitis.
Clostridia
Spore forming bacteria that is causative agent of food poisoning
Clostridia
Clostridia can cause
- Gas gangrene
- Tetanus
- Food poisoning
- colitis (pseudomembranous)
Differential stains can stain ___________ inside bacterial cells as well as free spores (both seen as the color ______), to identify these pathogenic bacteria.
endospores
green
can stain endospores inside bacterial cells as well as free spores (both seen as the color green)
Differential stains
What color do differential stains turn endospores and free spores?
green
The _________ of an organism is increased by the presence of a capsule, since the capsule protects the organism from phagocytosis.
virulence
If a bacterial infection is not being cleared or responding to antibiotic therapy as expected, staining of isolated organisms to determine the presence of a capsule may be warranted.
Gram negative bacteria that form capsules include
1.Haemophilus influenzae
2.Klebsiella pneumoniae
Gram positive bacteria that form capsules include
1.Bacillus anthracis
2.Streptococcus pneumoniae.
Identify the type of respiration you are considering for this test (pg 203)
Aerobic
There is a chemical reaction you are testing that is show in your lab book that states “Organisms capable of producing catalase rapidly degrade hydrogen peroxide….”
Identify the by-products that microbes produce.
water and free oxygen
Name the enzyme you are testing for that breaks down hydrogen peroxide.
Catalase
All microbes contain the enzyme you typed in question 4.
False
Why is this test (Catalase test) quick and easy with the increasing worry of resistant strains?
This test helps distinguish between catalase-positive staphylococci and catalase-negative streptococci, and members of enterobacteriaceae .
Most strains of MRSA make _______ and the catalase test can help differentiate between Staphylococci, which are typically catalase-positive, and streptococci which are typically catalase-negative. MRSA stands for Methicillin Resistant Staphylococcus Aureus.
catalase
T o F The matching test can be used to differentiate between catalase-positive Staphylococci and catalase-negative Streptococci.
False
Which carbohydrate is present at lower amounts to permit detection of the utilization of this substrate only?
Glucose
The TSI agar test is designed to differentiate among groups or genera of the
Enterobacteriaceae
Enterobacteriaceae, which are gram ________ _______
negative bacilli
Enterobacteriaceae, which are gram negative bacilli capable of fermenting _______ with the production of acid
glucose
Enterobacteriaceae, which are gram negative bacilli capable of fermenting glucose with the production of ______
acid
Purpose of TSI agar test
- differentiate Enterobacteriaceae
- distinguish from other gram negative intestinal bacilli
to distinguish Enterobacteriaceae from other _____ _______ _______ _______
gram negative intestinal bacilli
gram negative bacilli capable of fermenting glucose with the production of acid
Enterobacteriaceae
The TSI test can distinguish between 3 species of Proteus:
P. vulgaris,
P. mirabilis,
P. penneri.
These microorganisms are opportunistic pathogens, meaning that they do not normally cause disease, but can if they have the opportunity to colonize certain regions of the body.
P. vulgaris, P. mirabilis, and P. penneri.
This test (TSI) differentiates between these three microbes by testing their abilities to
reduce sulfur and ferment carbohydrates.
P. mirabilis causes
UTIS
P. mirabilis causes UTIS and can be treated with
ampicillin and cephalosporins.
more commonly causes nosocomial (hospital-acquired) infections in immunocompromised patients.
P. vulgaris
P. vulgaris more commonly causes
nosocomial (hospital-acquired) infections in immunocompromised patients.
Proteus less likely to cause UTIs
P. vulgaris
Proteus not sensitive to ampicilin and cephalosporins
P. vulgaris
Microbial growth is directly dependent on how temperature affects _______ ________.
cellular enzymes
Bacteria, as a group of living organisms, are capable of growth within an overall temperature range of
-5C to 80C (23F to 176F).
Each species, however, requires a narrower range that is determined by the ______ ________ of its enzyme systems.
heat sensitivity
minimum growth temperature
the lowest temperature at which growth will occur. Below this temperature, enzyme activity is inhibited and the cells are metabolically inactive so that growth is negligible or absent.
the lowest temperature at which growth will occur. Below this temperature, enzyme activity is _______ and the cells are metabolically inactive so that growth is negligible or absent.
inhibited
below minimum growth temperature enzyme activity is inhibited and cells are metabolically inactive, meaning
growth is negligible or absent
maximum growth temperature
the highest temperature at which growth will occur.
Above maximum growth temperature, most enzymes are_____
destroyed
cause of enzymes being destroyed
organism dies
optimum growth temperature
the temperature at which the rate of reproduction is most rapid
optimum growth temperature - the temperature at which the rate of reproduction is most rapid; however, it is not necessarily optimum or ideal for ____ enzymatic activities of the cell.
all
optimum growth temperature is not ideal for all
enzymatic activities of the cell
which major group would you likely find the microbes that reside in the human body?
mesophiles
This microbe is listed as being cold resistant; doubling its growth every 36 hours even at refrigerator temperatures or lower:
Listeria monocytogenes
Select two microbes below that would be examples of microbes surviving outside of the pH defense mechanisms of the body and causing infection.
H. pylori & C. albicans
Require oxygen. Growth is usually seen at the very top of a test tube.
Aerobes
Oxygen in excess blocks growth. In test tubes, growth is seen hovering away from, but close to, the surface.
microaerophiles
Lacks enzymes to break down free oxygen, so it is lethal. Growth is seen at the very bottom of test tubes.
obligate anaerobes
Can tolerate oxygen but show evenly dispersed growth in a test tube.
Aerotolerant anaerobes
Majority of bacteria can grow in the absence or presence of oxygen. Growth is seen congested near the surface but also spread throughout the test tube.
Facultative anaerobes
The causative agent of ___ _______is Clostridium perfringens.
gas gangrene
What is the causative agent of gas gangrene?
Clostridium perfringens
This bacteria thrives in wounds deprived of circulation and oxygen
Clostridium perfringens
The causative agent of gas gangrene is Clostridium perfringens. This bacteria thrives in wounds deprived of _______ and ________ which can cause limb loss and death
circulation and oxygen
Doctors may prescribe therapy using enriched oxygen delivered to the patient in a hyperbaric chamber allowing blood to carry more oxygen to the wounds, slowing the growth of _________microbes.
anaerobic
Cells are increasing in size.
However, no cell division = no increase in numbers.
Lag phase
Binary fission causes rapid increase in population.
Log phase
Cells dividing = cells dying.
No increase in cell number. Population at max.
Nutrients are depleting.
Toxic end products are accumulating.
stationary phase
Correct: Decrease of population at a rapid and uniform rate.
Resistant organisms usually persist after majority of population dies.
decline phase, death
stages of a growth curve in order
Lag
Log
Stationary
Death
How well did you create a lawn and how do you know?
A well created lawn of microbes would be densely populated except for the zones of inhibition. The bacterial colonies should be close together and cover the entire petri dish.
Are you looking for colonies on the dish? Compare it to a streak plate. What are you looking for
You are looking for zones of inhibition on the streak plate. There should be blank spots with no bacteria surrounding the antibiotic discs. This activity is different from a streak plate because the bacterial colonies are closely packed together to create a lawn.
What is a zone of inhibition?
A zone of inhibition tests a compound’s potency against a bacterial strain. It is a clear area surrounding some antimicrobial discs that indicate prevented growth of bacteria.
chemical substances used to treat infectious diseases.
chemotherapeutic agents
are synthesized and secreted by some true bacteria, actinomycetes, and fungi that destroy or inhibit the growth of other microorganisms.
Antibiotics
To determine a therapeutic drug of choice, it is important to determine its ___ ___ ______, possible adverse side effects in the host, and the scope of its antimicrobial activity.
mode of action
To determine a therapeutic drug of choice, it is important to determine its mode of action, possible adverse _____ ______ in the host, and the scope of its antimicrobial activity.
Side effects
To determine a therapeutic drug of choice, it is important to determine its
-mode of action
-side effects
-scope of antimicrobial activity
What is the essential tool used in clinical laboratories to select for the best agent?
Kirby Bauer Antibiotic sensitivity Test
How big was the petri dish that was made for this Harvard experiment? feet by feet
2x4
The bands of agar had antibiotic in differing concentrations toward the middle, what was the highest concentration of antibiotic?
1000x
It is expected for the bacteria to grow in the first outer sections of antibiotic bands, why?
The concentration of antibiotic is lower in the outer regions. Lower concentration of antibiotic means that there will be less substances that can kill the bacteria on the outside regions.
Why did bacteria grow in the second set of antibiotic bands?
Some of the colonies mutated and developed resistance to the higher concentration of antibiotic.
How much time passed before the bacteria reached the middle?
11 days
Bacteria which are normally ________ to an antibiotic can evolve resistance to extremely high concentrations in a short period of time.
sensitive
Bacteria which are normally sensitive to an antibiotic can _______ __________to extremely high concentrations in a short period of time.
evolve resistance
The efficiency of all disinfectants and antiseptics is influenced by a variety of factors including
1.Concentration
2.Length of exposure
3.Type of microbial population to be destroyed
4.Environmental conditions
Environmental conditions can include
1.Temperature
2.pH
3.Type of material on which the microorganisms exist
Numerous laboratory procedures are available for evaluating the antimicrobial efficiency of disinfectants or antiseptics. These procedures provide a _________ rather than an absolute measure of the effectiveness of any agent
general
These procedures provide a general rather than an absolute measure of the effectiveness of any agent because test conditions frequently _______considerably from those seen during practical use.
differ
Numerous laboratory procedures are available for evaluating the antimicrobial efficiency of disinfectants or antiseptics. These procedures provide a general rather than an absolute measure of the effectiveness of any agent because
test conditions frequently differ considerably from those seen during practical use.
What does MRSA (pronounced merrssa) stand for
Methicillin- resistant staphylococcus aureus
Which agent is named as a connection with resistance to MRSA?
A lack of susceptibility to benzalkonium chloride and other disinfectants
The size of the zone of inhibition is not indicative of the degree of effectiveness of the chemical agent. T o F
True
Which agent is named as a connection with resistance to MRSA? What’s the fix?
A lack of susceptibility to benzalkonium chloride and other disinfectants. Adjusted contact times might be needed to adequately kill the troublesome strains
Disinfectants and antiseptics are effective in reducing the number of living microbes. However, some microbes are capable of entering a dormant stage through a process called __________
sporulation.
In Sporulation, the microbes enclose themselves in structures that allow them to
preserve their genetic material
However, some microbes are capable of entering a dormant stage through a process called sporulation. In this stage, the microbes enclose themselves in structures that allow them to preserve their genetic material when environmental conditions are unfavorable, called _______.
dormancy
microbes enclose themselves in structures that allow them to preserve their genetic material when environmental conditions are
unfavorable
sporulation. In this stage, the microbes enclose themselves in structures that allow them to preserve their genetic material when environmental conditions are unfavorable, called
dormancy
called dormancy. For example, a bacterial cell that has encapsulated itself and entered a dormant stage is called an _________
endospore
Endospores are a type of reproductive egg that bacteria produce in order to survive unfavorable conditions. T o F
False
Makes it easier to distinguish colonies of the desired organism from other colonies on the plate
Differential media
Favors the growth of a particular microorganism. Used for preliminary isolation.
enrichment culture
designed to suppress the growth of unwanted bacteria and encourage the growth of the desired microbes
selective media
The area of no bacterial growth around an antimicrobial agent in the disk-diffusion method
zone of inhibition
The common name for a bacterium in the family Enterobacteriaceae.
enterics
Aerobic or facultatively anaerobic, gram-negative, non–endospore-forming, rod-shaped bacteria that ferment lactose with acid and gas formation within 48 hours at 35°C.
coliforms
coliforms
Aerobic or facultatively anaerobic, gram-negative, non–endospore-forming, rod-shaped bacteria that ferment lactose with acid and gas formation within 48 hours at 35°C.
