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I am working on gram positive bacteria for which OD was reported and some reported mcfarland value. I am new to microbiology. i want to confirm the only thing that matters is cfu. please reply for what shoul I prefer thank you.
ONPG Test – Principle, Procedure, Uses and Interpretation
The ability of bacteria to ferment lactose depends on two enzymes, permease and beta-galactosidase. Permease allows lactose to enter the bacterial cell wall, where it is then broken down into glucose and galactose by beta-galactosidase. The glucose and galactose can then be metabolized by the bacteria. However, some organisms lack permease and appear as late or non-lactose-fermenters.
The ONPG test is considered to be a very sensitive test for lactose-fermentation. O-nitrophenyl-beta-D-galactopyranoside (ONPG), an artificial substrate, is incorporated into this test and acts as the substrate for the beta-galactosidase to ascertain the particular enzyme activity which subsequently aids in the identification and differentiation of different organisms.
What is Primary Cell Culture?
Primary cell culture is the disassociation of cells from a parental animal or plant tissue through enzymatic or mechanical measures and maintaining the growth of cells in a suitable substrate in glass or plastic containers under controlled environmental conditions. Cells in primary culture have the same karyotype (number and appearance of chromosomes in the nucleus of a eukaryotic cell) as those cells in the original tissue. Primary cell culture could be classified into two based on the kind of cells used in culture.
- Anchorage Dependent or Adherent Cell – These cells require an attachment for growth. Adherent cells are usually derived from tissues of organs, for instance from kidney where the cells are immobile and embedded in connective tissue.
- Anchorage Independent or Suspension Cells – These cells do not require an attachment for growth. In other words, these cells do not attach to the surface of culture vessel. All suspension cultures are derived from cells of the blood system for instance, white blood cell lymphocyte is suspended in plasma.
Cells derived from primary cultures have a limited life span. Cells cannot be held indefinitely due to several reasons. Increasing cell numbers in primary culture will lead to exhaustion of substrate and nutrients. Also, cellular activity will gradually increase the level of toxic metabolites in the culture inhibiting further cell growth.
At this stage, a secondary or a subculture has to be performed to ensure continuous cell growth.
- At least three to five well-isolated colonies of the same morphological type of both test and control strains are selected from an agar plate culture. The top of each colony is touched with a loop, and the growth is transferred into a tube containing 4 to 5 ml of a suitable broth medium, such as tryptic soy broth.
- The broth culture is incubated at 37°C until it achieves or exceeds the turbidity of the 0.5 McFarland standard (usually 2 to 6 hours)
- The turbidity of the actively growing broth culture is adjusted with sterile saline or broth to obtain turbidity optically comparable to that of the 0.5 McFarland standard. This results in a suspension containing approximately 1 to 2 x 10 8 CFU/ml for E.coli ATCC 25922. To perform this step properly, either a photometric device can be used or, if done visually, adequate light is needed to visually compare the inoculum tube and the 0.5 McFarland standard against a card with a white background and contrasting black lines.
- Optimally, within 15 minutes after adjusting the turbidity of the inoculum and control strains suspension, sterile cotton swabs are dipped into each of the adjusted suspensions. The swabs should be rotated several times and pressed firmly on the inside wall of the tube above the fluid level. This will remove excess inoculum from the swab.
- A dried Müeller-Hinton agar plate is divided into 3 halves.
- The dried surface of a Müeller-Hinton agar plate is inoculated by streaking the control strains evenly across the upper and lower thirds of the plate, and the test strains between the control, leaving a distance of not more than 5mm on each side of the control strain.
- Allow the inocula to dry for few minutes with the lid.
- Place antimicrobial discs in the gap between the test and control strain using a sterile forceps and press gently.
- Within 30 mins of applying the discs, incubate the plates aerobically at 35-37 °C for 18-24 hrs.
Open Source Spectrophotometer projects
Cambridge iGEM 2010 E.glometer
“We designed and built a low cost electronic system for measuring light output, this is useful for reporter assays.”
Aachen OD/F device
“Measuring Optical Density (OD) or absorbance is one of the indispensable elements in the field of microbiology. One question that has to be answered often is how many cells are in a suspension? Here, the OD can give a hint. However, the commercially available OD meters are expensive and limit its application and usage in low budget institutions.
Therefore, here we present our OD/F Device. The device is specifically designed for biohackspaces, Do It Yourself (DIY), community laboratories and schools. With our OD/F Device, we aim to enable precise and inexpensive scientific research.”
Definition of Bacterial Isolation
Bacterial isolation is defined as the technique of separating one species of bacteria from the bacteria’s mixed culture by different plating methods like pouring, spreading, streaking, and serial dilution. The growth of bacteria can be observed over the solid nutrient medium, in the liquid broth medium and some automated liquid culture medium. To visualize and isolate the bacteria in solid media, we need to add the bacterial suspension into or on the media.
Oppositely, the bacterial inoculum in the liquid broth is characterized by the turgid media. The automated liquid culture medium also detects bacteria’s presence through various characteristics like production of carbon dioxide. Therefore, bacterial isolation provides an important tool to study the morphological, physiochemical and pathogenic properties of the bacteria that has been isolated.
Madeleine Leininger is considered as the founder of the theory of transcultural nursing.
Her theory has now developed as a discipline in nursing.
Evolution of her theory can be understood from her books:
- Culture Care Diversity and Universality (1991)
- Transcultural Nursing (1995)
- Transcultural Nursing (2002)
Transcultural nursing theory is also known as Culture Care theory.
Theoretical framework is depicted in her model called the Sunrise Model (1997).
ABOUT THE THEORIST
One of the first nursing theorist and transcultural global nursing consultant.
MSN - Catholic University in Washington DC.
PhD in anthropology - University of Washington.
She developed the concept of transcultural nursing and the ethnonursing research model.
For more details: http://en.wikipedia.org/wiki/Madeleine_Leininger
Transcultural nursing is a comparative study of cultures to understand similarities (culture universal) and difference (culture-specific) across human groups (Leininger, 1991).
Set of values, beliefs and traditions, that are held by a specific group of people and handed down from generation to generation.
Culture is also beliefs, habits, likes, dislikes, customs and rituals learn from one’s family.
Culture is the learned, shared and transmitted values, beliefs, norms and life way practices of a particular group that guide thinking, decisions, and actions in patterned ways.
Culture is learned by each generation through both formal and informal life experiences.
Language is primary through means of transmitting culture.
The practices of particular culture often arise because of the group's social and physical environment.
Culture practice and beliefs are adapted over time but they mainly remain constant as long as they satisfy needs.
Is a set of belief in a divine or super human power (or powers) to be obeyed and worshipped as the creator and ruler of the universe.
refers to a group of people who share a common and distinctive culture and who are members of a specific group.
a consciousness of belonging to a group.
the sense of being part of an ethnic group or culture
commonalities of values, norms of behavior, and life patterns that are similar among different cultures.
values, beliefs, and patterns of behavior that tend to be unique to a designate culture.
refers to objects (dress, art, religious arti1acts)
refers to beliefs customs, languages, social institutions.
composed of people who have a distinct identity but are related to a larger cultural group.
a person who crosses two cultures, lifestyles, and sets of values.
refers to the fact or state of being different. Diversity can occur between cultures and within a cultural group.
People of a minority group tend to assume the attitudes, values, beliefs, find practices of the dominant society resulting in a blended cultural pattern.
the state of being disoriented or unable to respond to a different cultural environment because of its sudden strangeness, unfamiliarity, and incompatibility to the stranger's perceptions and expectations at is differentiated from others by symbolic markers (cultures, biology, territory, religion).
share a common social and cultural heritage that is passed on to successive generations.,
refers to a subjective perspective of the person's heritage and to a sense of belonging to a group that is distinguishable from other groups.
the classification of people according to shared biologic characteristics, genetic markers, or features. Not all people of the same race have the same culture.
It is an in-depth self-examination of one's own background, recognizing biases and prejudices and assumptions about other people.
Culturally congruent care
Care that fits the people's valued life patterns and set of meanings -which is generated from the people themselves, rather than based on predetermined criteria.
Culturally competent care
is the ability of the practitioner to bridge cultural gaps in caring, work with cultural differences and enable clients and families to achieve meaningful and supportive caring.
Leininger (1991) identified three nursing decision and action modes to achieve culturally congruent care.
Cultural preservation or maintenance.
Cultural care accommodation or negotiation.
Cultural care repatterning or restructuring.
MAJOR CONCEPTS [Leininger (1991)]
Illness and wellness are shaped by a various factors including perception and coping skills, as well as the social level of the patient.
Cultural competence is an important component of nursing.
Culture influences all spheres of human life. It defines health, illness, and the search for relief from disease or distress.
Religious and Cultural knowledge is an important ingredient in health care.
The health concepts held by many cultural groups may result in people choosing not to seek modern medical treatment procedures.
Health care provider need to be flexible in the design of programs, policies, and services to meet the needs and concerns of the culturally diverse population, groups that are likely to be encountered.
Most cases of lay illness have multiple causalities and may require several different approaches to diagnosis, treatment, and cure including folk and Western medical interventions..
The use of traditional or alternate models of health care delivery is widely varied and may come into conflict with Western models of health care practice.
Culture guides behavior into acceptable ways for the people in a specific group as such culture originates and develops within the social structure through inter personal interactions.
For a nurse to successfully provide care for a client of a different cultural or ethnic to background, effective intercultural communication must take place.
APPLICATION TO NURSING
To develop understanding, respect and appreciation for the individuality and diversity of patients beliefs, values, spirituality and culture regarding illness, its meaning, cause, treatment, and outcome.
To encourage in developing and maintaining a program of physical, emotional and spiritual self-care introduce therapies such as ayurveda and pancha karma.
HEALTH PRACTICES IN DIFFERENT CULTURES
Use of Protective Objects
Protective objects can be worn or carried or hung in the home- charms worn on a string or chain around the neck, wrist, or waist to protect the wearer from the evil eye or evil spirits.
Use of Substances .
It is believed that certian food substances can be ingested to prevent illness.
E.g. eating raw garlic or onion to prevent illness or wear them on the body or hang them in the home.
Burning of candles, rituals of redemption etc..
The use of folk or traditional medicine is seen among people from all walks of life and cultural ethnic back ground.
Within a given community, specific people are known to have the power to heal.
Immigrant groups have their own cultural attitudes ranging beliefs and practices regarding these areas.
In many cultures, the male is dominant figure and often they take decisions related to health practices and treatment. In some other cultures females are dominant.
In some cultures, women are discriminated in providing proper treatment for illness.
Beliefs about mental health
Mental illnesses are caused by a lack of harmony of emotions or by evil spirits.
Problems in this life are most likely related to transgressions committed in a past life.
Factors such as unemployment, underemployment, homelessness, lack of health insurance poverty prevent people from entering the health care system.
It is varies for different cultures groups.
Respect the client's personal space when performing nursing procedures.
The nurse should also welcome visiting members of the family and extended family.
NURSING PROCESS AND ROLE OF NURSE
Determine the client's cultural heritage and language skills.
Determine if any of his health beliefs relate to the cause of the illness or to the problem.
Collect information that any home remedies the person is taking to treat the symptoms.
Nurses should evaluate their attitudes toward ethnic nursing care.
Understand the influence of culture, race &ethnicity on the development of social emotional relationship, child rearing practices & attitude toward health.
Collect information about the socioeconomic status of the family and its influence on their health promotion and wellness
Identifiy the religious practices of the family and their influence on health promotion belief in families.
Understanding of the general characteristics of the major ethnic groups, but always individualize care.
The nursing diagnosis for clients should include potential problems in their interaction with the health care system and problems involving the effects of culture.
The planning and implementation of nursing interventions should be adapted as much as possible to the client's cultural background.
Evaluation should include the nurse's self-evaluation of attitudes and emotions toward providing nursing care to clients from diverse sociocultural backgrounds.
Self-evaluation by the nurse is crucial as he or she increases skills for interaction. .
Nurses need to be aware of and sensitive to the cultural needs of clients.
The practice of nursing today demands that the nurse identify and meet the cultural needs of diverse groups, understand the social and cultural reality of the client, family, and community, develop expertise to implement culturally acceptable strategies to provide nursing care, and identify and use resources acceptable to the client (Andrews & Boyle, 2002).
Murphy SC. Mapping the literature of transcultural nursing. J Med Libr Assoc . 2006 Apr94(2 Suppl):E143-51.
Leninger M. Culture Care Theory: A Major Contribution to Advance Transcultural Nursing Knowledge and PracticesJournal of Transcultural Nursing, Vol. 13 No. 3, July 2002 189-192.
Leininger M. Culture care diversity and universality: A theory of
nursing. New York: National League for Nursing Pres 1991.
Leininger M.Transcultural nursing: Concepts, theories, research,
and practice. Columbus, OH: McGraw-Hill College Custom Series 1995.
Andrews MM, Boyle JS.Transcultural concepts in nursing care. J Transcult Nurs. 2002 Jul13(3):178-80.
George Julia B. Nursing theories: The base of professional nursing practice 5rd edition. Norwalk, CN: Appleton and Lange 2002.
Kozier B, Erb G, Barman A, Synder AJ. Fundamentals of nursing concepts, process and practice, Edn 7th, 2001.
Leninger M, McFarland M. Transcultural Nursing: Concepts, Theory, Research, and Practice Edn 3rd, McGraw-Hill Professional New York, 2002.
Assay Protocol to Measure Cytotoxicity
Additional Reagents Required:
- Culture medium, e.g., RPMI 1640 (R0883) containing 10% heat inactivated FCS (fetal calf serum, 12106C), 2 mM glutamine (G6392) and 1 μg/ml actinomycin C1 (actinomycin D, A9415).
- If an antibiotic is to be used, additionally supplement media with penicillin/streptomycin or gentamicin
- Tumor necrosis factor-α, human (hTNF-α) (10 μg/ml), sterile (T6674).
For the determination of the cytotoxic effect of human tumor necrosis factor-α (hTNF-α, T6674) on WEHI-164 cells (mouse fibrosarcoma, 87022501) (Figure 2).
- Preincubate WEHI-164 cells at a concentration of 1 × 10 6 cells/ml in culture medium with 1 μg/ ml actinomycin C1 for 3 h at 37 °C and 5-6.5% CO2.
- Seed cells at a concentration of 5 × 10 4 cells/ well in 100 μl culture medium containing 1 μg/ml actinomycin C1 and various amounts of hTNF-α (final concentration e.g., 0.001–0.5 ng/ml) into microplates (tissue culture grade, 96 wells, flat bottom).
- Incubate cell cultures for 24 h at +37 °C and 5-6.5% CO2.
- After the incubation period, add 10 μl of the MTT labeling reagent (final concentration 0.5 mg/ml) to each well.
- Incubate the microplate for 4 h in a humidified atmosphere (e.g., +37 °C, 5-6.5% CO2).
- Add 100 μl of the Solubilization solution into each well.
- Allow the plate to stand overnight in the incubator in a humidified atmosphere (e.g., +37 °C, 5-6.5% CO2).
- Check for complete solubilization of the purple formazan crystals and measure the absorbance of the samples using a microplate (ELISA) reader. The wavelength to measure absorbance of the formazan product is between 550 and 600 nm according to the filters available for the ELISA reader, used. The reference wavelength should be more than 650 nm.
Figure 2. Determination of the cytotoxic activity of recombinant human TNF-α (hTNF-α) on WEHI-164 cells (mouse fibrosarcoma) using MTT assay.
Minimum Bactericidal Concentration (MBC) Assay
Emery Pharma routinely provides antibiotic susceptibility testing for the following methods according to Clinical and Laboratory Standards Institute (CLSI) guidelines. For more information, see our MIC guide in the resources section:
- broth micro- and macro-dilution (see illustration below)
- disk diffusion
- agar dilution
Test articles can be natural or synthetic, mixtures or purified. Hundreds of bacterial strains , including multi drug-resistant clinical isolates, ESKAPE pathogens, and multiple fungal strains , are available in our inventory for immediate testing.
MIC and MBC Assays. To set up an MIC/MBC assay, (1) first prepare 2-fold serial dilutions of the test compounds (up to 7) and one quality control (QC) antibiotic in a microdilution plate. (2) Create the inoculum by taking a few colonies from an agar plate with a sterile swab, preparing a McFarland standard, and diluting the McFarland standard into media. (3) Dispense the inoculum into the microdilution plate with the serial diluted test compounds and incubate the microdilution plate. (4) Read the microdilution plate to determine the MIC value. (5) Plate a portion of each well on an appropriate agar media, incubate the agar, and check for colonies to determine the MBC.
Interpretation of Microdilution Results. Depicted here is a typical MIC assay conducted according to CLSI microdilution guidelines. Up to 7 compounds and one quality control (QC) antibiotic are serially diluted from column 1 to column 11 of a 96-well microplate to form a concentration gradient. Column 12 serves as a positive growth control. In the illustration, “no growth” is represented by white circles and “growth” is represented by yellow circles. The MIC value is the lowest concentration of a compound/antibiotic at which no growth is observed.
CLSI M100 Performance Standards for Antimicrobial Susceptibility Testing Approved Standard-28 th edition January 2018
CLSI M07 Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically Approved Standard-11 th edition January 2018.
CLSI M02 Performance Standards for Antimicrobial Disk Susceptibility Tests Approved Standard-13 th edition January 2018.
CLSI M27 Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts Approved Standard-4 th edition November 2017.
CLSI M38 Reference Method for Broth Dilution Antifungal Susceptibility Testing of Filamentous fungi Approved Standard-3 rd edition November 2017.
Click on the image below to watch a video of our Biology scientists explaining the basics of the MIC assay!
Red Fluorescent Protein (RFP)
Red fluorescent protein (RFP) is a versatile biological marker for monitoring physiological processes, visualizing protein localization, and detecting transgenic expression in vivo. RFP can be excited by the 488 nm or 532 nm laser line and is optimally detected at 588 nm.
We offer a series of Invitrogen CellLight RFP fusion constructs of signal peptides or cell structure proteins with tagRFP for accurate and specific targeting to subcellular structures, including the cytoskeleton, mitochondria, and secretory compartments.
To complement these tools, we also offer anti-RFP antibodies and antibody conjugates for a wide variety of applications, including imaging, western blotting, and flow cytometry. All of our anti-RFP antibodies are suited for detection of native RFP, RFP variants, and CellLight fusion proteins.