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Bacterial Transformation Efficiency: E.Coli with pGLO

Bacterial Transformation Efficiency in E.Coli with pGLO Plasmids
By: Richard Stone
Introduction “The conversion of one genotype into another by the introduction of exogenous DNA (that is, bits of DNA from an external source) is termed transformation. The transformation was discovered in Streptococcus pneumoniae in 1928 by Frederick Griffith; in 1944, Oswald T. Avery, Colin M. MacLeod, and Maclyn McCarty demonstrated that the “transforming principle” was DNA. Both results are milestones in the elucidation of the molecular nature of genes.” 1
Bacteria transformation is the process of a bacteria absorbing and expressing foreign genetic information using plasmids. Plasmids are small circular molecules of DNA that holds a small number of genes. The plasmids used in the experiment have the ampicillin resistance gene. Ampicillin (amp) is an antibiotic used to kill bacteria such as E. coli, the bacteria used in the experiment. E. coli (Escherichia coli) is a simple bacterium commonly found in our body’s and in everyday life but most commonly found in mammal’s intestines. Glowing Fluorescent Proteins (GFP’s) is the gene found in jellyfish that holds bioluminescent properties and “glow” under UV light. By knowing the location of the gene, scientists can “cut out” the GFP gene from the jellyfish DNA. They do this using restriction enzymes to which recognize and cut DNA in a specific region of nucleotides to acquire a specific gene. Once the gene is isolated, it can be used in the experiment and “glued” into a plasmid that contains the AMP gene. This is done by sticky ends as the Jellyfish DNA binds to the amp resistance plasmid using hydrogen bonds which are hen sealed by DNA ligase. This creates pGLO a plasmid which is used in the experiment in the transformation of the bacteria. Before it can be part of the transformation the bacteria must be made competent to accept the pGLO. This is done by “heat shocking” the bacteria which makes it easier for the pGLO to be incorporated into the bacteria. For the bacteria to fluoresce sunder UV light it must be in presence of arabinose sugars, which “turns on the gene for the production of Glowing Fluorescent Proteins.2 The amp Resistance gene enables bacteria to survive in the presence of the antibiotic ampicillin. When a plasmid containing both the GFP gene and AMP gene (pGLO) is transferred into an E. coli bacterium, the transformed cells can be grown in a culture dish that contains ampicillin. Only a small number of bacteria cells will be transformed and grow on the LB (lysogeny broth) and amp plates and glow. 3
The experiment demonstrates how Bacteria is modified to express a specific gene through the process of bacterial transformation. The purpose of this experiment is to find the efficiency of bacterial transformation in E. Coli bacteria by observing their expression of the plasmids. This is calculated by determining the frequency of the bacterium with GFP’s and arabinose sugars by counting the glowing colonies.
It was the results for each plate was hypothesized before the experiment. The LB plate with only the bacteria and no pGLO administered will grow a lawn of bacteria and have no glowing properties. The LB with ampicillin but bacteria without pGLO will not survive at all and there will be no bacteria growth. The LB plate with amp and bacteria with the pGLO will have bioluminescent properties but only a very small percentage of the bacteria will survive the amp and bacterial transformation will occur. Finally, the LB with no amp but the bacteria with the pGLO will form a lawn of bacteria and the bacteria that is transformed will glow like the previous plate. The efficiency of the bacterial transformation is hypothesized using in class discussion and background knowledge, to be about 8×10-4 %. 4
Materials and Methods E. coli bacteria cultures
100-1000 µl micropipette
0.5-10 µl micropipette
sterile tips
2 sterile 15-ml test tubes
500 μL of ice cold 0.05M CaCl2 (ph. 6.1)
500 μL of lysogeny broth/agar
a spreading rod
Bunsen burner
4 agar plates: 2 ampicillins and 2 ampicillin –
an incubator
a sterile inoculating loop
10 μL of pAMP solution
a timer
sterile glass beads
a water bath
1. Use a permanent marker to label one sterile 15-ml tube “ ”, and another “-“.
2. Use a 100-1000 µl micropipette and sterile tip to add 250 µl of CaCl2 (calcium chloride) solution to each tube.
3. Place both tubes on ice.
4. Use a sterile inoculating loop to transfer a visible mass of E. coli from a starter plate to the tube:
a. Sterilize loop in Bunsen burner flame until it glows red hot.
b. Carefully, stab loop into agar to cool.
c. Scrape up a visible mass of E. coli, but be careful not to transfer any agar. (Impurities in agar can inhibit transformation.)
d. Immerse loop tip in CaCl2 solution and vigorously tap against the wall of the tube to dislodge bacteria. Hold tube up to light to observe the bacteria drop off into the calcium chloride solution. Make sure cell mass is not left on a loop or on side of tube.
e. Sterilize loop before setting it on the lab bench.
5. Immediately suspend cells in the tube by repeatedly pipetting in and out, using a 100-1000 µl micropipette with a fresh sterile tip.a. Pipet carefully to avoid making bubbles in suspension or splashing suspension far up sides of the tube.
b. Hold tube up to light to check that suspension is homogeneous. No visible clumps of cells should remain.
6. Return tube to ice.
7. Transfer the second mass of cells to – tube as described in Step 4, and resuspend cells as described in Step 5.
8. Return – tube to ice. Both tubes should be on the ice.
9. Use a 0.5-10 µl micropipette to add 10 µl of 0.005 µg/µl pGFP solution directly into cell suspension in the tube. Tap tube with a finger to mix. Avoid making bubbles in suspension or splashing suspension up to the sides of the tube. [DO NOT ADD pGFP TO THE “-” TUBE.]
10. Return tube to ice. Incubate both tubes on ice for 15 minutes.
11. While cells are incubating, use a permanent marker to label two LB plates and two LB/amp plates with name and the date.
Label one LB/amp plate “ GFP”. This is the experimental plate.
Label the other LB/amp plate “- GFP”. This is a negative control.
Label one LB plate “ GFP”. This is a positive control.
Label the other LB plate “- GFP”. This is a negative control.
12. Following the 15-minute incubation on ice, heat shock the cells in both the and – tubes. It is critical that cells receive a sharp and distinct shock:
a. Carry ice beaker to the water bath. Remove tubes from ice, and immediately immerse in 42°C water bath for 90 seconds.
b. Immediately return both tubes to ice, and let stand on ice for at least 1 additional minute.
13. Place and – tubes in test tube rack at room temperature.
14. Use a 100-1000 µl micropipette with a fresh sterile tip to add 250 µl of sterile LB medium to each tube. Gently tap tubes to mix. This will allow the cells to recover from the heat shock.
15. Use the matrix below as a checklist as and – cells are spread on each plate:
16. Use a 100-1000 µl micropipette with a fresh sterile tip to add 100 µl of cell suspension from the – tube onto the – LB plate and another 100 µl onto the – LB/amp plate.
17. Use a 100-1000 µl micropipette with a fresh sterile tip to add 100 µl of cell suspension from the tube onto LB plate and another 100 µl of cell suspension onto LB/amp plate. [Do not let suspensions sit on plates too long before proceeding to Step 18.]
18. Use sterile glass beads to spread cells over the surface of each – plate:
a.Obtain four 1.5 ml tubes containing at least five sterilized glass beads.
b.Lift lid of one – plate, only enough to allow pouring of the beads from one of the 1.5 ml tubes onto the surface of the agar. Replace plate lid; do not set the lid down on the lab bench. Repeat for all plates.
c.Use beads to spread bacteria evenly on plates by moving plates side to side several times. Do not move plates in a circular motion.
d.Rotate plates ¼ turn, and repeat spreading motion. Repeat two more times. The object is to separate cells on agar so that each gives rise to a distinct colony of clones.
19. Let plates set for several minutes to allowing the suspension to become absorbed into the agar. Then wrap together with tape.
20. Place plates upside down in 37°C incubator, and incubate for 12-24 hours, or store at room temperature for approximately 48 hours.5
Results Transformed cells
Non-transformed cells
Bacterial Growth in form of green colonies
No growth on plate
Growth spread across entire plate (bacteria lawn)
Growth spread across entire plate (bacteria lawn)
Table 1. the E. coli bacterial plates after incubation.
Discussion Before the experiment was conducted the results of each plate was hypothesized. It was believed that the plate with only the LB and no plasmids added would grow a lawn of bacteria, this was proven correct by the experiment. The plate with LB and ampicillin but no pGLO was predicted to have no growth, which was also proven correct by the experiment. The plate with LB and ampicillin but the bacteria was administered with the pGLO was predicted to survive the amp but not in very large quantities. Finally, for the plate with only LB but with the pGLO administered to the bacteria it was hypothesized that it would glow, not necessarily in large quantities but at least a little. This was different from the results of the experiment in which the bacteria did not show bioluminescent properties. This can occur for numerous reasons, the lack of bacteria that was transformed, unsterile equipment, improper heat shocking to make the bacteria competent. While all these are the possible reasoning for the experiment results the most probable cause for the plates to not grow is the lack of arabinose sugar which is an important part in the expression of the GFP’S (see introduction). If the plates lack the arabinose sugar the GFP proteins may not be expressed. This explains why the LB only plate with the pGLO did not produce transformed bacteria. This also draws questions to why the plate with LB and ampicillin and the transformed bacteria. Why would it glow if it didn’t have any arabinose sugar? This most likely is explained by the fact that it must have been administered in the LB but not in the others.3
The transformation Efficiency was determined by counting the number of colonies on the LB/amp plate pGFP. Any bacteria that shows light under the UV light must have accepted the plasmids and successfully transformed the desired genes to survive the lb/amp plate and express the GFP gene. Each colony represents one bacteria that has been transformed. Using this the efficiency can be determined. Transformation efficiency is expressed as the number of antibiotic resistant colonies per µg of pGFP DNA. To find this the mass of the pGFP used must first be determined by the formula Concentration X Volume = Mass. This is shown in figure 1 and was calculated using the formula 0.005 µg /µl x 10 µl = 0.05 µg. Then using the formula to determine the total number of cells per plate the fraction of cells suspended onto the LB/Amp plate. This is shown in figure 1 and was calculated using the formula .005 µg/510 µl=9.8×10^-5 µg /µl this number must them be multiplied by 100 because there are approximately 100 cells in use. This is calculated in figure 1 and is solved to be 9.8×10^-3. To determine the transformants per microgram the formula (total transformed cells/cells per plate)/10000 to find the efficiency in transformants per microgram. This is solved in figure 1 to be 8.673 transformants per microgram. Then the Transformation Efficiency can be found. This is shown in figure 1 which uses the formula (Total cells to start / total microliters) x 100 microliters to find the total number of cells on the plate. Then the formula (Transformants/ Total cells) x100 = percent of efficiency. This is calculated as (8.673 transformants/ 1,960,784,314) x100 to calculate a transformation efficiency of .000004335% or in scientific notation 10x 4.3355 ^ -6
Before conducting the experiment, it was hypothesized that the transformation efficiency would be about 8×10^-4%. After doing the experiment the transformation efficiency was found to be 4.335×10^-6% or 8.673 transformants per microgram. This proves the percentage of efficiency to be significantly lower than hypothesized. The transformation efficiency being lower than expected shows the rarity of this specific form of genetic modification. The experiment tests how rare it is for the genetic modification to occur and demonstrates the results of the modification and its effect on an organism.
Citations Griffiths, Anthony JF. “Bacterial Transformation.” An Introduction to Genetic Analysis. 7th Edition. U.S. National Library of Medicine, 01 Jan. 1970. Web. 31 Dec. 2016.
“Bacterial Transformation.” SpringerReference (n.d.): n. pag. Cold Spring Harbor Laboratory. Dolan DNA Learning Center. Web.
Reece, Jane B. Campbell Biology, Volume 1. Boston, MA: Peason Learning Solutions, 2011. Print. Chapter 20
Transfer, Genetics, And Information. BIOTECHNOLOGY: BACTERIAL TRANSFORMATION* (n.d.): n. pag. Web
“Lab Center – Bacterial Transformation.” Lab Center – Bacterial Transformation. N.p., n.d. Web. 03 Jan. 2017.
“Bacterial Transformation.” SpringerReference (n.d.): n. pag. Web.

Postoperative Nausea and Vomiting: Causes and Treatments

Postoperative nausea and vomiting is the nausea and vomiting symptoms which occurred after a surgery, medicines intake or anaesthesia usage. Around 18 to 30 of surgical patients have PONV and the nausea and vomiting symptoms are usually self-limiting in most cases.1 Uncomplicated PONV usually resolve within 24 hours after an operation whereas intractable PONV involve various triggering factors and resist to medical treatment, making it harder to treat. Studies revealed that most patients dislike chronic PONV more than postoperative pain as it is a more distressing illness and it may lead to several serious clinical consequences if left untreated.
In the case of repeated vomiting, PONV patients might suffer from dehydration and have a higher chance of developing hiatal hernia, a condition where the upper part of stomach protrudes into the thorax through the opening of diaphragm. Other than that, patients might also experience anorexia, gastrointestinal discomfort, headache, weakness, dizziness and nausea while not vomiting. Chronic vomiting can also cause complications like dental damage and sore throats due to exposure of oesophageal lining and mouth cavity to the low pH gastric acid. Moreover, PONV may induce serious problems like pulmonary aspiration, electrolyte abnormalities, wound dehiscence, increased pain and oesophageal rupture.4,5 Despite causing patients discomfort, patients also have to pay more for the delayed hospital discharge. Each incidence of vomiting has increased postanaesthetic care unit (PACU) stay duration by 20mins. Therefore, to reduce the unanticipated hospital admission and the financial burden brought by PONV, there is a need to understand the disease pathophysiology so that precise and mechanism-based treatment strategies can be developed to tackle the emesis problem.
The vomiting centre and the chemoreceptor trigger zone (CTZ) are the two main parts of the brain controlling the vomiting action. The vomiting centre is located within the medulla oblongata and the emesis action is initiated via the stimulation of five primary afferent pathways. They are the chemoreceptor trigger zone, vagal mucosal pathway of the gastrointestinal system, neuronal pathways from the vestibular apparatus system, inputs from the periphery glossopharyngeal nerve and reflex afferent pathways from cerebral cortex C2,3 and midbrain afferents. Next, efferent nerve impulses are sent to various place of the body such as the pharynx, larynx, diaphragm, intercostals muscles and gut to initiate the vomiting reflex. During the ejection phase of the vomiting reflex, the diaphragm and abdominal muscles simultaneously contract and the elevated intra-abdominal pressure leads to the throw up and expulsion of gastric contents. A variety of receptors are participated in the emesis action. They are the histaminergic(H1), dopaminergic(D2), serotonergic(5-HT3), muscarinic and neurokinin-1 receptors. Consequently, pharmacological agents which target on these receptors can be utilized to treat PONV. However, the British National Formulary (BNF) had advised that antiemetic agents should only be used once the causative factor for nausea and vomiting was identified. This is because the use of antiemetic is sometimes dangerous and inappropriate in clinical cases like diabetic ketoacidosis, digoxin or antiepileptic overdose.6 Hence, the aetiology and possible causative factors of PONV should be investigated to guide the planning of the pharmaceutical management steps and the antiemetic selection for treating PONV.
There are patient-specific factors, surgical factors and anaesthetic risk factors which contribute to PONV prevalence. Patients who aged 6 to 16 year old, female, non-smoker, obese or have a history of motion sickness or PONV are proven to be the high-risk patient group. Moreover, patients who have chemotherapy, migraine and gastroparesis problems are also susceptible to PONV. Other causative factors include elevated intracranial pressure, metabolic abnormalities, gastroduodenal ulcers, dehydration and infections of the gastroesophageal lining.
As for the surgical factors, PONV is related to the premedication side-effect, prolong fasting, conditions of gastric inflation during mask ventilation, use of long-acting opioids, nitrous oxide, volatile anaesthetics and high dose neostigmine in surgery. In addition, frequent head movement of patient and early intake of food after surgery can also potentiate the nausea problem.1 Some types of operations have higher chance of developing PONV, they are the gynaecological surgery, ear, nose and throat operation, intra-abdominal and squint correction surgery. Furthermore, the surgical duration is also an important contributor which predisposes patients to a higher risk of PONV. Every 30 minutes extension in surgical time can increase risk of PONV by 60% as patient is taking in more anaesthetics into the body. Hence, healthcare team should control and minimize the surgery duration such that risk of getting PONV is reduced.
Although it is not relevant to discuss anaesthetic techniques in this case scenario, it is important to note that regional anaesthesia should be preferred over general anaesthesia during surgical process. According to SOGC guideline, there is an 11-fold increase in the PONV risk when using general anaesthesia rather than regional anaesthesia. Apart from that, volatile anaesthesia, long-acting opioid and neostigmine should also avoid in surgery as these agents predispose patient to PONV. If the use of general anaesthesia is unavoidable in a surgery, propofol can be a suitable induction agent because it induces less PONV incidence.
A thorough assessment should be carried out to serve as a rationale for the management plan of PONV. The past medical history, frequency and nature of the vomiting episode, blood electrolyte test and physical examination can be evaluated to identify the severity of disease condition and the aetiology of PONV. Subsequently, the appropriate pharmacological agents which target on the responsible pathway of emesis can be given.
Many antiemetic preparations are available in the market and patients can choose between formulations of solution, buccal tablets, rectal suppository and subcutaneous (SC), intravenous (IV) or intramuscular (IM) injections when oral route is not feasible.6 As no single agent provides complete control in emesis, most hospital has adopted a multimodal approach and a combination strategy where different antiemetics which target on different receptors are utilized in the treatment of PONV.1 Combination therapy becomes the preferable way to treat PONV and the generally used combination is 5-HT3 receptor antagonists with droperidol or dexamethasone.
Granisetron and ondansetron are examples of 5-HT3 or serotonin receptor antagonists. They exert their effects in the chemoreceptor trigger zone and at vagal afferents of the gastrointestinal tract. Previous studies showed that no single agent performed exceptionally well than the others of same class as all 5-HT3 antagonists illustrated similar safety and efficacy profile. Yet, a recent meta-analysis which includes 85 randomized controlled, double-blind studies with 15,269 patients involvement had established that the antiemetic effect of granisetron is significantly superior to ondansetron and dolasetron. Ondansetron was also found to be more cost effective than granisetron. 1-2mg of granisetron or 4-8mg of ondansetron can be delivered in intravascular route at the end of surgery for PONV treatment. Long-acting serotonin antagonist with higher binding affinity to 5-HT3 receptors, palonosetron, is also available in the market with a long half-life of about 40 hours. Patients receiving these agents might experience headache, constipation and dizziness problems.
Droperidol is a butyrophenone which acts competitively on central dopaminergic receptors in the chemoreceptor trigger zone (CTZ). It is applied in 0.625-1.25mg IV route at the end of surgery. A systematic review of 24 randomized studies was carried out by Schaub and team, they concluded that droperidol decreases PONV incidence regardless of the dose given to patients. However, this drug is only used as a third-line antiemetic for intractable PONV when other alternative treatments failed because droperidol can lead to adverse effects associated with QT prolongation and torsades de points, sedation, anxiety, hypotension and extrapyramidal symptoms. Due to its possibility in causing fatal arrhythmia, electrocardiographic monitoring is compulsory each time upon its usage. Nonetheless, a double-blinded randomized clinical study which included 120 patients stated that there was insufficient evidence to prove the QTc prolongation effect induced by droperidol after surgery.
Dexamethasone is classified under corticosteroids and often delivered in a 4 to 5mg ‘one-off’ dose via IV or IM route.19 The exact mechanism of action is unknown but it is related to the peripheral inhibition of prostaglandin synthesis and its ability to reduce 5-HT turnover in the CNS. Although dexamethasone is not licensed for the indication of PONV, this drug is as effective as other conventional antiemetic drugs like droperidol and serotonin antagonists. A single blinded, randomized-controlled interventional study had illustrated that the administration of dexamethasone is useful for the reduction of PONV episodes (30% in contrast to 70% of the placebo group).20 Moreover, Ormel et al. illustrated that the addition of dexamethasone to droperidol and ondansetron showed a profound amplification in the efficacy profile of these triple agents combination. It stands as a good alternative for PONV treatment due to the advantage of cost-effectiveness issue and its characteristic of long action duration. As dexamethasone can increase plasma glucose level, it is not recommended for diabetic patient. Furthermore, unfavorable side-effect like postoperative euphoria, impaired wound healing, irritability and adrenal suppression can happen in patient taking long-term corticosteroids.
Metoclopramide is a gastroprokinetic agent which acts on the D2 receptors of the gastrointestinal tract. It can accelerate the gastric emptying rate of gastroparesis and GI obstruction patients.2,6 Despite blocking the D2 receptors, it also has antagonist action on 5-HT3 receptors in the CTZ and vomiting centre when delivered in high doses. 5 to 20mg dose of metoclopramide in subcutaneous, oral or IV route is commonly taken by patient before meal and before bed.6 This medicine is commonly administered as combination therapy because there is conflicting evidence stating that metoclopramide alone is ineffective for PONV and it should not be use unless the causative factor for PONV is gastric stasis. Yet, a recent meta-analysis has proved that 10mg IV metoclopramide does well in preventing nausea and vomiting problems after the general anaesthesia surgery. As with the phenothiazines discussed below, both drugs have limited use in practice due to the adverse reactions like extrapyramidal effects and dystonia disorder particularly in pediatric and young adults population.
Phenothiazines is an example of strong dopamine antagonist which also act on medullary CTZ. Promethazine, prochlorperazine and perphenazine belong to this group and take part in the prophylaxis and treatment of PONV.24 Prochlorperazine is often administered as a 12.5mg deep intramuscular injection or in a 3 to 6mg dose buccal preparation 12 hourly after the surgery. These agents show superior efficacy in treating opioid-induced PONV. However, high-dose metoclopramide and phenothiazines are now less likely used in clinical practice because of their significant side effects like acute dystonic reactions, sedation, dizziness and extrapyramidal symptoms.9,25 A systematic analysis consisting of 19 non-randomized and randomized clinical trials had demonstrated that most studies supported the effectiveness of promethazine in reducing PONV occurrence when compared to placebo and that combination therapy is always preferable and more effective than promethazine alone.
Cyclizine is an antihistamine drugs which block the H1 sympathetic pathway in the vomiting centre. The antimuscarinic and antihistamine properties of cyclizine render it to become an antiemetic drug in PONV treatment. A randomised double-blinded study which involved the participations of 960 women had shown that patients who received cyclizine monotherapy showed a slightly greater antiemetic effect than granisetron alone (PONV incidence of 24% with cyclizine compare to 23% in granisetron group).26 Cyclizine can be given orally, intramuscularly or intravenously, with common antimuscarinic side-effects like sedation and dry mouth. Severe heart failure patient should avoid taking this medicine because it leads to detrimental haemodynamic effect.6 The acidic pH of cyclizine at 3.2 also causes pain and irritancy to body upon injection.10 As a result, patients usually have 50mg of cyclizine IV injection every 8 hours after proper dilution. A lower dose of 25mg in oral, IM or IV preparations can also be applied in elderly patient.
Scopolamine has anticholinergic property which inhibits the muscarinic as well as the histaminergic receptors in the vestibular apparatus and the nucleus of the tractus solitarus.3,9 Patients who undergo middle ear surgery or use opioids as postoperative anaesthetics are recommended to take scopolamine for their profound efficacy in reducing PONV.3 Scopolamine requires 2 to 4 hours for onset of duration. Hence, a fast-acting antiemetic or a loading bolus dose is needed in urgent case. It is available in transdermal form as a 1.5mg patch which can be placed behind the ear. This slow-release formulation can have sustained effect up to 72 hours. Apfel C et al. had reported that transdermal scopolamine had significantly reduced the risk of PONV when compared to the placebo group although it has the main side-effects of dry mouth, sedation and visual disturbances.28 Furthermore, a comparative study between the combination use of ondansetron plus scopolamine patch and ondansetron alone also proved that the earlier group significantly decrease the nausea and vomiting incidence after surgery.
Other than a mechanism-based approach, less conventional therapeutic agents can also be used to treat intractable PONV cases. An antidepressant with a novel indication, mirtazapine, is able to ease the nausea and vomiting symptoms because it can antagonize 5-HT3 receptors. A small scale randomized trial which compared the therapeutic outcome of mirtazapine and ondansetron had showed that patients using mirtazapine were less anxious and had fewer PONV episodes than the ondansetron group. Next, olanzapine which is recognized as an atypical antipsychotic drug also proved to have potential in treating PONV. It can inhibit several receptors such as the dopamine, acetylcholine, histamine and 5-HT3 receptors. Ibrahim M et al. had conducted a randomized controlled study which involved 82 surgical patients. The result proved the efficacy and safety profile of olanzapine against PONV especially during the late postoperative stage. Other than medications approach, non-pharmacological interventions also show potential therapeutic efficacy in PONV management.
Acupuncture, acustimulation or acupressure serves as a good alternative or adjuvant therapy for PONV patients as it shows good tolerability and safety profile. The P6 point (Neiguan) which located at 5cm near to the ventral wrist is the target site of these alternative approaches. Transcutaneous electrical stimulation delivered to the P6 point of the pericardium meridian has been proved to be an efficient way in preventing emesis. Patients only complain of light side-effects like needle fainting, allergy, needle site pain, anxiety or lethargy problems when using this method.
In order to solve the labour intensive and time-consuming issues of traditional Chinese acupuncture, the acupressure and acustimulation wristband are introduced in the market (Sea-Band and ReliefBand). Sea-Band applies steady, continuous pressure on the P6 point whereas ReliefBand is a watch-like device which conducts low current to P6 point via electrodes in contact with the skin. Based on the well-established efficacy profile and good evidence-base literature support, healthcare professionals can involve more acupuncture interventions in treatment practice as part of the multimodal approach.
In this case, the intractable emesis symptoms experienced by the old woman might indicate the failure of prophylaxis treatment or the need to start a primary antiemetic treatment. Before the initiation of a rescue treatment, a bedside examination and a patient interview should be done to find out whether the PONV symptom is associated to issues such as morphine analgesia, surgical pain management, infection, intestinal obstruction, hypotension, hypoxia, blood in the pharynx, anxiety or removal and insertion of nasogastric tube.
5-HT3 antagonist is the recommended drug for patients who previously do not receive a prophylaxis treatment. Patient can start with a low dose regimen such as ondansetron 1 mg, dolasetron 12.5 mg and granisetron 0.1 mg. If drugs for prophylaxis had been given but fail, patients can then try other class of antiemetics to tackle more diverse receptor pathways. For instance, Habib et al. had found that the failure of prophylactic ondansetron or droperidol can be replaced with rescue agents like promethazine (12.5-25 mg IV), prochloperazine (12.5mg IM) or cyclizine (25-50mg IV or IM) to achieve a better outcome. This is because consensus guideline support that the repeat use of 5-HT3 antagonist within the initial 6 hours postoperative period provides no extra recovery response. If patient use dexamethasone as prevention agent, small dose 5-HT3 antagonist (25% of prophylactic dose) can then be given as a rescue approach. A study also concluded that the cost-effectiveness of ondansetron in low dose treatment group was higher than that in the high dose prophylatic group.
Moreover, in the case of the aggressive treatment failure, such as those who had taken 5-HT3 antagonist, droperidol and dexamethasone altogether but failed, repeat dosing of same prophylactic regimen except dexamethasone can only be considered 6 hours after the surgery though the optimal dosage and timing for readministration still remain unknown. Transdermal scopolamine can also be prescribed for outpatients as it is a more convenient preparation than the parenteral drugs.
Prolong use of opioids for pain control after surgery should also be minimized as side-effects like nausea and vomiting are correlated to the prescribed dose. Alternative analgesics like NSAIDS can be used to substitute the causative opioids. In persisting case, pharmacist can review the prescription and anaesthetic charts to ensure adequate maintenance of analgesia, antiemetic and oxygen supply. Dose escalation under safety and therapeutic dosage range can also be worked on. However, pharmacist should be cautious on polypharmacy problem as it may aggravate nausea and vomiting in susceptible patient. Non-oral drug preparations can be considered over oral route to avoid burdening of patient with excessive pills at one time. If necessary, the acupuncture treatment can also be applied to attempt a multimodal approach.
Pharmacist should also concern about the possible dehydration risk that might be encountered by chronic PONV patients. For this reason, the blood pressure, hydration and perfusion level of patients have to be checked on a regular basis. Patients should be told to report of symptoms like dry or sticky mouth, sunken eyes, reduced urination or dark yellow urine. If constipation or diarrhea happens, intravenous fluid replacement therapy, osmotic or stimulant laxative can be given to solve the issues. For the dietary measures, patients should avoid oily or spicy food which might aggravate the nausea. Small, frequent meal is preferable over big heavy meal as light meal reduce the possibility of gastric discomfort.
Patients should be advised to not move around too often to avoid triggering the vomiting centre. Furthermore, in post-discharged nausea and vomiting (PDNV) case, the antiemetic efficacy profiles are different from PONV’s as they have dissimilar underlying cause. Droperidol should be avoided as it is ineffective in treating PDNV.2 If the patient still not responsive to all these approaches, specialist intervention should be initiated to treat intractable nausea and vomiting symptoms. Serious causative factors like surgical complication might be suspected and further investigations are required to treat this disease.
In a nutshell, the optimization management of PONV disease requires the participation of the multimodal approach. Patients should be treated accordingly after the accurate disease assessment and further modifications of treatment approaches like (dose-adjustment, introduction of new agents or alternative approaches) can be done to control patient’s nausea and vomiting symptoms. Lifestyle modification and non-pharmacological interventions also play an important part in treating PONV. Proper patient education about symptoms management should be delivered and follow-up session can be arranged to assess patient’s rehabilitation progress. Apart from that, reassurance and full supportive care from healthcare teams also play an important role in reducing patient distress and anxiety level.